CN118510388A

CN118510388A - Improved screening methods for genome editing events

Info

Publication number: CN118510388A
Application number: CN202280085177.9A
Authority: CN
Inventors: T·J·戈尔兹; D·德弗莱斯豪韦尔; E·博西尔; K·德哈卢因; F·莫伊勒韦特
Original assignee: BASF Agricultural Solutions Seed US LLC
Current assignee: BASF Agricultural Solutions Seed US LLC
Priority date: 2021-12-22
Filing date: 2022-12-20
Publication date: 2024-08-16
Also published as: WO2023118136A1; AU2022422286A1; CA3240580A1

Abstract

The present invention is in the field of plant molecular biology and relates to a method for improved screening against known edits within the genome of a cell. The methods of the invention include dividing a population of plant cells comprising a desired nucleic acid sequence into subgroups, quantifying the concentration of the desired nucleic acid sequence in each subgroup, culturing cells from the one or more subgroups, dividing the cells into subgroups, quantifying the concentration of the desired nucleic acid sequence in each subgroup, and regenerating an intact individual plant from cells of one or more selected subgroups having the desired nucleic acid sequence.

Description

Improved screening methods for genome editing events

Technical Field

The present invention is in the field of molecular biology, in particular plant molecular biology, and relates to methods for improved screening against known edits within the genome of a cell (e.g. as a direct result of genome editing).

Background

If cells carrying a particular genomic mutation do not contain a label that allows for direct detection of the cells, it may be difficult to detect and enrich them. Such mutations may occur naturally or spontaneously, for example during growth and breeding processes, due to exogenous or endogenous DNA damage, or due to genetic methods or genome editing processes.

Genome editing techniques, including the introduction of precise gene editing, are well documented in plants. Methods well established in the art use techniques such as the following to introduce double-stranded DNA breaks (DSBs) in the genome: zinc finger nucleases, homing endonucleases, TALENs or RNA-guided nucleases (e.g., CRISPR systems (e.g., cas lambda) or CRISPR CAS systems (e.g., CRISPR CAS9 or CRISPR CAS a)).

If Double Strand Breaks (DSBs) in the genome are repaired by an error-prone non-homologous end joining pathway (NHEJ), genome editing applied to plant cells results quite effectively in mutations comprising targeted insertions and/or deletions (InDel), or in case the editing method is unsuccessful, it results in an unaltered genome sequence. If the DSBs are repaired by homologous recombination and repair or donor templates, precise Editing (PE) can be achieved, albeit at a much lower frequency.

Although the efficiency of production of cells comprising targeted editing has been improved since the development of CRISPR methods compared to the use of homing endonucleases or TALENs, it remains a relatively rare event in eukaryotic cells, especially without the introduction of a selectable marker gene that enables direct selection/enrichment of mutated or edited cells.

Zhang et al (Zhang et al 2016, nat. Commun. [ Nature. Communication ] 7:12617) describe two methods of genome editing in which CRISPR/Cas9 is introduced as DNA or RNA into plant cells and transiently expressed using a transgene-free protocol, resulting in edited genes. This procedure avoids antibiotic/herbicide selection. After CRISPR/Cas9DNA or RNA is introduced into wheat immature embryos by particle bombardment and transiently expressed, callus cells are generated and plants are regenerated. T0 plants were examined by PCR-RE and DNA sequencing to identify targeted mutants. The absence of the herbicide selection step supports the generation of transgenic-free mutants even after bombardment with CRISPR/Cas9 DNA. Plants with targeted mutations and lacking active transgenes were obtained using both methods (transiently expressed CRISPR/Cas9DNA and CRISPR/Cas9 RNA). In addition, liang et al (Liang et al, 2018,Nature Protocols [ Nature. Scheme ], volume 13, phase 2, page 413 f) describe a DNA-free editing method in which Ribonucleoprotein (RNP) of CRISPR/Cas9 is delivered into target cells by particle bombardment. This protocol allows for the production and identification of edited plants within nine to eleven weeks without the use of any exogenous DNA and without the antibiotic/herbicide selection step.

Both Zhang et al (Zhang et al 2016, nat. Commun. [ Nature. Communication ] 7:12617) and Liang et al (Liang et al, 2018,Nature Protocols [ Nature. Protocol ], vol.13, 2 nd, p.413 f) utilized mutant screening methods using a pool of shoots (pool). Labeled leaf sample pieces of three to four shoots produced from the same bombarded embryo were pooled and then checked for editing events by PCR-RE. If desired, shoots in pools with positive PCR-RE signals were allowed to grow for several more days and then checked by PCR-RE alone and confirmed by sequencing. However, a relatively low frequency of mutagenesis was observed in the plants analyzed.

Furthermore, WO 2018/001884 provides a method that supports the preparation, selection and propagation of organisms containing specific predetermined mutations by a combination and resolution method, since the statistical probability is very low when reference is made to searching for predetermined nucleotide substitutions. WO 2018/001884 describes a method in which a pool of mutated organisms is first divided into sub-pools while maintaining the reproductive potential of organisms from each genotype within the analysis pool, and then the presence of mutations is tested by PCR (e.g., ddPCR). The organisms of the positive pool are then subsequently divided into secondary pools, and then the pool is retested for the presence of mutations in each genotype by PCR (e.g., ddPCR) while again retaining the reproductive potential of the organisms from each genotype within the analysis pool. Thus, the method requires that each genotype be maintained within each sub-pool, such that the reproductive potential of organisms from each genotype within the analysis pool is maintained. Thus, the method includes, for example, repeating propagation of all existing genotypes, and then analyzing at least one copy of each genotype in order to maintain another copy of each genotype in the population. Thus, the population produced by the method of WO 2018/001884 contained copies of the mutagenized genotype, whereas its presence was identified via a digital PCR-based system after the propagation step.

The methods disclosed in WO 2018001884, zhang et al and Liang et al for identifying positive clones are expensive and cumbersome.

Thus, there remains a need in the art for methods that allow for the efficient and rapid identification of those cells that contain the desired mutation (e.g., mutation at the target site) after the mutagenesis step. There is a need for a method that allows identification of those organisms that display the desired nucleotide sequence (positive event) in a known region in a large population of organisms.

Disclosure of Invention

The present invention thus relates to a three-step method for producing a plant comprising a desired nucleic acid sequence, the method comprising or preferably consisting of the following selection steps:

1. Dividing a population of cells comprising regenerative cells into subsets, testing a sample from each subset to quantify the concentration of the desired nucleic acid sequence,

2. Culturing cells from one or more subgroups having the highest concentration of the desired nucleic acid sequences, dividing the cultured cells (e.g., recovered callus or shoots) into subgroups as tested in a sample of the subgroup, and dividing the cultured cells into subgroups, and testing the concentration of the desired nucleic acid sequences in a sample from the subgroup; and

3. Regenerating a plant from the cultured cells (e.g., callus or shoots), preferably producing a plant from callus or shoots of a subset of cells having the highest concentration of the desired nucleic acid sequence, and testing for the presence of the desired nucleic acid sequence in the plant, selecting a plant from the plants tested, wherein the selected plant comprises the desired nucleic acid sequence.

The term "desired nucleic acid sequence" shall mean the sequence of a nucleic acid molecule that is known in an organism and that is searched for as presented in the methods of the invention. Such desired sequences may be the result of desired modifications of the preferred natural form of the natural, undesired nucleic acid molecule or nucleic acid sequence present in a plurality of variants. The desired modification may be, for example, a variation or modification of the sequence compared to a control or wild type. Modifications may be preferred natural variations of the sequence, or may be the result of a genome editing process or mutagenesis procedure.

The term "desired nucleic acid sequence (desired nucleic acid sequence)" shall also include "desired nucleic acid sequence (desired nucleic acid sequences)". The term "desired nucleic acid sequence" shall mean that a nucleic acid molecule has more than one known and searched sequence. The term "desired nucleic acid sequence" shall also mean that more than one nucleic acid molecule has a known and searched sequence. Thus, the term "desired nucleic acid sequence" also includes a plurality of modifications to one or several nucleic acid molecules to form the desired nucleic acid sequence. Thus, the desired nucleic acid sequence may also comprise several modifications in different positions of the nucleic acid molecule, and/or several modifications in different nucleic acid molecules, e.g. modifications in the genome.

For example, the profile of modifications can be found on more than one molecule of the genome. For example, modifications defining a "desired nucleic acid sequence" may be found in one or more alleles, in one or more genes, or on one or more chromosomes of an organism. Preferably, the presence of a modification, part or all thereof, can be identified by a rapid and reliable method as described herein, e.g., a quantitative PCR method, preferably ddPCR (digital droplet PCR), or a high throughput sequencing method, like Next Generation Sequencing (NGS).

The term subgroup with the highest concentration of "desired nucleic acid sequence" means that the concentration of the desired nucleic acid sequence in the respective subgroup is higher than in the other subgroups, preferably highest.

The concentration of the desired nucleic acid sequence may be determined, for example, by measuring or determining the copy number of the desired nucleic acid sequence in the sample. For example, the concentration per volume, amount, or other quantifiable amount of a quantitative method (e.g., ddPCR reaction) may be determined. There are other methods known to those skilled in the art that will allow for accurate quantification of a desired nucleic acid sequence in a sample.

Thus, in one embodiment, the invention includes a method for producing a plant comprising a desired nucleic acid sequence, the method of the invention comprising the steps of:

a) Dividing a plant cell population comprising regenerated plant cells into subgroups, the plant cell population comprising a subpopulation of cells comprising a desired nucleic acid sequence, quantifying the concentration of the desired nucleic acid sequence for each subgroup, each subgroup representing a subset of the genotype of the population, and identifying one or more subgroups having the highest concentration of the desired nucleic acid sequence,

B) Culturing cells from one or more subgroups having the highest concentration of the desired nucleic acid sequences, dividing the cells into subgroups, quantifying the concentration of the desired nucleic acid sequences in each subgroup, each subgroup representing a subset of the genotype of the population, and selecting one or more subgroups having the highest concentration of the desired nucleic acid sequences, and

C) Regenerating an intact individual plant from the cells of the one or more selected subsets of step (c) having the desired nucleic acid sequence.

In contrast to prior art methods, the methods of the present invention do not require treatment of the cells being tested, so that the reproductive potential of organisms from each genotype within the analytical cell is maintained. It has been found that even if the genotype pool is modified as a result of the examination, it is sufficient to determine the concentration of the desired sequence. Thus, according to the invention, each subgroup and each sample does not represent all genotypes present in the pool.

The regenerative cells used in the method of the invention are individual plant cells or cells within plant explants. In the methods of the invention, substantially all explant types capable of producing dividing cells to produce multicellular calli capable of plant regeneration can be used.

According to the invention, a subset of cells identified in step (a) as comprising a suitably high concentration of the desired nucleic acid sequence is selected and the cells are cultured to proliferate the cells and, depending on the rejuvenated cells used in the method of the invention, rejuvenated cells.

Thus, according to the method of the invention, "culturing the cells" means maintaining the cultured cells under conditions that allow the cells to proliferate, preferably starting the regeneration process to produce tissue from which shoots and plants can be recovered. Thus, "culturing cells" includes proliferating the cultured cells. The step of "culturing the cells" may comprise morphogenic development of the cells, for example to form, for example, somatic embryos or shoots from a subset of the cells selected in step (a). Thus, "cultured cells" include cells that are cultured that can regenerate into callus or shoots. For example, a "cultured cell" can include a process that proliferates cells (e.g., selected cells) and enriches cells (e.g., callus). Thus, in one embodiment of the invention, the cultured cells are calli that divide and enrich them.

Thus, in a preferred embodiment, depending on the regenerating cells used in the method of the invention, the "cultured cells" in step (b) of the method of the invention means the step of regenerating the cells selected in culturing step (a) to produce plant tissue or plants.

Plant cells comprising regenerative cells are for example selected from the group consisting of:

single cells, such as protoplasts or microspores,

Cell aggregates, such as cell suspensions or callus cultures,

Complex multicellular explants from mature or immature seeds, such as immature embryos, scutellum or cotyledons,

Complex multicellular explants from seedlings, such as roots, hypocotyls, cotyledons, leaves, petioles or meristems, and

Complex multicellular explants from plants, such as roots, leaves, leaf bases, petioles, stems or meristems.

Thus, preferably, the cells are selected from the group consisting of: protoplasts, microspores, cell suspensions, callus cultures, immature embryos, scutellum, cotyledons, roots, leaves, leaf bases, petioles, stems, meristems, roots, leaves, leaf bases, petioles, stems and meristems.

The plant cells comprising regenerative cells used in the method of the present invention are not limited to these examples.

There is great interest in developing efficient methods for recovering and growing plants derived from genetically modified cells. Typically, only those cells that contain the desired nucleic acid sequence at the target location can be selected by analyzing the nucleic acid sequence at the desired location, for example, via PCR or nucleic acid sequencing. These methods require extraction and isolation of RNA and/or DNA of the modified cells, a process that damages the relevant cells. Thus, the methods known in the art require that the mutagenized cells must first be cloned or propagated before the presence of the desired nucleic acid sequence is analyzed. Thus, in the methods of the prior art (WO 2018001884), in order to preserve or confirm the presence of each genotype in a viable population for plant regeneration, the presence of the desired nucleic acid sequence is determined by a cell destructive method for extracting nucleic acid molecules (e.g., DNA and/or RNA) of cells in a sample of the cloned or proliferated cell population. Plants can be regenerated from the remaining cell population comprising cells having the genotype identified in the analysis.

However, in accordance with the present invention, it has unexpectedly been found that it is sufficient to obtain a sample of a cell population without cloning or proliferating cells. For example, it has been unexpectedly found that cloning or propagation steps as shown in the art (e.g., WO 2018001884) can be skipped. In the methods of the invention, it is not necessary to first clone the regenerating cells to ensure that a sufficient number of the desired genetic modifications are found in the regenerated plant or plant part. Advantageously, identifying the concentration of the desired nucleic acid sequence in a sample taken directly from a population of regenerative cells (which should contain the desired nucleic acid sequence of, for example, a genome-edited or genetically modified regenerative cell) allows for efficient production of plants carrying a nucleic acid molecule having the desired nucleic acid sequence.

Thus, in one embodiment, a population of plant cells comprising regenerative cells is first divided into subgroups and then the concentration of the desired nucleic acid sequences in each subgroup is tested in said subgroup.

Thus, the method of the invention allows for the much faster, more efficient production of plants comprising the desired nucleic acid sequence from regenerative cells, as it is performed without the time consuming steps of cloning and proliferating genetically modified (e.g., mutagenized, engineered or edited) regenerative cells.

In one embodiment, plant cells comprising regenerative cells allow plant regeneration.

In one embodiment, the plant cell comprising the regenerative cell comprises a genetically modified regenerative cell. Thus, in one embodiment, the methods of the invention comprise genetically modifying nucleic acid molecules of a cell, and then determining the concentration of a desired nucleic acid sequence in a subset of the modified cells.

Thus, in one embodiment, the method of the invention comprises a step wherein the nucleic acid molecules or genomes of plant cells comprising the regenerating cells are chemically mutated or ionized, genetically or genomically edited, or genetically engineered prior to the division of the cells into subsets. In another embodiment, the plant cells comprising the regenerative cells are genetically modified after a subset of regenerative cells has been formed, e.g., as described herein. For example, genetic modifications are made separately in each subgroup.

A "genetically modified" cell refers to a cell that is the target of a treatment that modifies or alters its nucleic acid sequence (e.g., modifies one or more bases in its genome or modifies the sequence of a nucleic acid molecule of the genome, resulting in modification of the cell's DNA). The term "genetically modifying" a cell refers to a method of effecting a genetic modification in one or more cells. According to the invention, the nucleic acid sequence or genome of a cell may be genetically modified, for example by mutagenesis via treatment with chemicals or by irradiation, oligonucleotide-directed mutagenesis (ODM), RNA interference, recombinant DNA methods or genome or genetic engineering or editing (e.g. using site-directed nuclease (SDN)). Thus, the term "genetically modified cell" shall also mean a "mutated cell" or a "mutagenized cell" or a "genome-edited cell". The term "genetically modified" shall also include modification or mutation of the nucleic acid sequence or genome of a cell at a predefined site. Oligonucleotide-directed mutagenesis (ODM) allows modification of a nucleic acid sequence (e.g., genome) of a cell without producing the mutation in an unpredictable manner.

"Genetic modification" also results from a method of transforming or transfecting a cell. In transformation or transfection of cells, additional nucleic acid molecules are introduced to delete a portion of the genome or to effect a change in the genomic sequence. Recombinant DNA methods include, for example, insertion of a selected nucleic acid molecule into the genome of a cell or deletion of a sequence via homologous recombination. Preferably, the regenerative cells are modified by genomic or genetic editing. Substantially all targeted genomic or gene editing methods can be used, ranging from NHEJ to HDR, e.g., base editing, lead editing, targeted random mutation techniques, and the like.

In a preferred embodiment, the method of genetically modifying the plant cell is targeted genomic modification or mutagenesis, such as mutation at one or more target sites, and includes, but is not limited to, single site mutation and targeted random mutation. In one embodiment, the genetic modification is an insertion or deletion, more preferably an insertion deletion or a point mutation, of one or more nucleic acids.

Thus, in one embodiment, the invention comprises the steps of:

-performing targeted genetic modification of plant cells comprising regenerated plant cells, then dividing the population of plant cells comprising regenerated plant cells into subgroups, and quantifying the concentration of the desired nucleic acid sequence of each subgroup.

Targeted modification may also be performed after the plant cell population comprising regenerated plant cells is divided into subgroups. Thus, in another embodiment, the invention comprises the steps of:

-after dividing the population of plant cells comprising regenerated plant cells into subgroups, performing targeted genetic modification on plant cells comprising regenerated plant cells, and quantifying the concentration of the desired nucleic acid sequence of each subgroup.

Methods for introducing mutations at predefined sites of a nucleic acid molecule in a cell are well known to those skilled in the art and include, for example, methods such as introducing targeted mutations, e.g., via use of meganucleases, homing endonucleases, zinc finger proteins, TALENs, oligonucleotides (e.g., PNA, DNA, etc.), and the like. In one embodiment, the mutation of the nucleic acid sequence of the cell used in the methods of the invention (e.g., to produce a callus, shoot, or plant) involves base editing or lead editing, e.g., using a CRISPR/Cas system. Thus, in one embodiment, the method comprises a step wherein a CRISPR (e.g., cas9 or Cas12a system) has been used to introduce a mutation at a target site.

Mutations can be generated by: introducing a corresponding enzyme (CRISPR component) into the cell, such as Ribonucleoprotein (RNP); or stably or transiently transforming a cell with one or more nucleic acid molecules encoding a corresponding protein or enzyme or nucleic acid molecule (e.g., a gRNA) or mediating expression of the corresponding molecule or mixture thereof. Thus, the method of the invention comprises introducing a mutation at a predefined site in the genome.

In one embodiment, the method comprises a step wherein a random mutation is introduced at or near the target site, preferably a targeted random mutation. Methods for generating allelic diversity are known to those skilled in the art and include, for example, methods based on site-specific recruitment of mutagenized proteins such as error-prone DNA polymerase or highly active DNA deaminase. The mutagenized protein sequences may be specifically delivered to the target site by fusion or non-covalent interactions with proteins such as programmable DNA binding domains and DNA endonucleases (e.g., CRISPR-Cas effector proteins). Thus, in one embodiment, the method comprises the step wherein DNA nucleotides within a defined genomic window are altered by a CRISPR-associated cytosine and/or adenine base editor. Adenine and cytosine base editors catalyze targeting a.t to g.c or c.g to t.a base pair changes, respectively. Cytosine base editors that can be used for targeted mutagenesis include targeted AID-mediated mutagenesis (Ma et al, 2016), CRISPR-X (Hess et al, 2016) and SunTag-based supermutans (Rees and liu, 2018). The double cytosine and adenine base editor system includes STEME (Li et al 2020), A & C-BEmax (Zhang et al 2020) and SPACE (Gru newald et al 2020). Other methods of introducing targeted random mutations include EvolvR and TRACE or similar methods: halperin, S.O. et al Nature [ Nature ]560,248-252 (2018); gru newald, J. Et al Nat Biotechnol [ Nature biotechnology ]38,861-864 (2020); rees, h.a. and Liu, d.r. (2018) Nature reviews, genetics [ natural review, genetics ],19 (12), 770-788; chen H et al Nat Biotechnol [ natural biotechnology ] month 2 2020; 38 (2) 165-168; zhang, X. Et al Nat Biotechnol [ Nature Biotechnology ]38,856-860 (2020); hess GT et al Nat Methods [ Nature method ] month 12 of 2016; 13 (12) 1036-1042; ma, Y. Et al Nat Methods [ Nature. Method ]13,1029-1035 (2016); li, C. Et al Nat Biotechnol [ Nature Biotechnology ]38,875-882 (2020).

Methods of producing mutated cells for use in the present invention may include steps describing the delivery of DNA or protein into the cell, such as bombardment in a gene gun delivery system or agrobacterium-mediated transformation. The method includes, for example, a step of single cell transformation of regenerative cells, such as multicellular explants (e.g., immature embryo explants) or single cells (e.g., protoplasts or microspores). Additional target cell types are mentioned herein and are known to those of skill in the art. The step of bombardment or agrobacterium-mediated transformation in the gene gun delivery system may be performed before or after the formation of the subgroups. Corresponding methods are known in the art or are described herein.

The methods of the invention are particularly useful if only a phenotypic or agronomic difference (e.g., in advanced stages of development or growth) between a genetically modified cell or derived plant or part thereof (e.g., a cell comprising a desired nucleic acid sequence in a target region) and a cell or derived plant or part thereof comprising a wild-type or undesired nucleic acid sequence can be detected with difficulty. In some cases, visualization may also depend on the number of target genes in the plant. Advantageously, the molecular screening method of the present invention is not dependent on any agronomic or phenotypic differences.

Furthermore, the present invention allows for rapid screening to identify cells and plants comprising a desired nucleic acid sequence without first selecting for cells or plants via selectable marker gene expression and selection methods. The use of selectable markers is well known to those skilled in the art and examples are described in the prior art. The presence of the desired nucleotide sequence at the target region cannot generally be identified by selection with a selection marker that provides for the readout of cells that have received the DNA component and may have a higher chance of carrying the mutation.

Furthermore, it is generally undesirable, if not impossible, to introduce additional nucleic acid molecules or sequences into cells. Thus, the methods of the invention comprise enriching for cells of interest without the use of external or endogenous selection markers (e.g., fluorescent reporters (such as GFP or BFP) or antibiotic or herbicide tolerance or resistance genes).

Thus, in one embodiment, the presence of the desired nucleic acid sequence exhibits any detectable effect at a relevant time during cultivation, development or growth, for example until at least 1,3, 6 or 9 months after modification of the regenerating cells, or only after the plants have begun to mature, or only when the plants are grown in the field or only when they are grown in a particular environment, such that the presence of the desired nucleic acid sequence can be assumed. In some cases, the presence of a desired nucleic acid sequence can only be detected under specific biotic or abiotic stress conditions (e.g., stress conditions such as, for example, drought, nitrogen deficiency, or chemical treatment). In this case, screening as in the method of the invention allows plants comprising the desired nucleic acid sequence to be produced earlier and less time consuming.

Thus, in one embodiment, the medium or growth conditions in which the cells, tissues or plants are cultured are not selective for the presence of the desired nucleic acid sequence in the genome of the cells or tissues or plants comprising the modified cells.

Thus, according to the invention, it is sufficient to (i) divide the modified cells (e.g., modified plant cells comprising regenerated plant cells) into subgroups, (ii) analyze the sample of each subgroup for the concentration of the desired nucleic acid sequence, and (ii) continue with the subgroup or subgroups having the highest concentration to produce a plant comprising cells comprising the desired nucleic acid sequence. The highest concentration of the desired nucleic acid sequence is preferably determined after modification of the plant cells comprising regenerated plant cells.

For each step of the method of the invention, the concentration of the desired nucleic acid sequence at the target site can be determined by a sensitive, quantitative, preferably rapid method. Thus, in one embodiment, the method of the invention comprises determining the concentration of the nucleic acid sequence in the genome of the genetically modified plant cell in a molecular sieve. The concentration of the desired sequence in the samples of the subgroup is tested. According to the invention, the concentration of the nucleic acid sequence in the sample can be determined by quantitative molecular analysis methods (e.g. by qPCR, preferably by ddPCR), allowing in subsequent sequential steps to select those subgroups comprising cells carrying the desired mutation. Quantitative molecular analysis methods were performed in samples taken from the subgroup.

Advantageously, the present invention allows for the generation and testing of a faster number of genetically modified regenerative cells, as no time consuming and resource binding steps are required for proliferation or cloning of regenerative cells after the modification step. Thus, the method of the invention comprises in step (a) of the invention directly testing the concentration of the desired nucleic acid sequence in a subset of plant cells comprising the genetically modified regenerative cells. Such testing occurs in samples taken from a subset.

According to the invention, for each step of the method of the invention, the term "identifying the subset or subsets having the highest concentration of the desired nucleic acid sequences present" thus means determining the concentration of the desired nucleic acid sequences in a sample of said subset. Preferably, the samples of the subgroup represent the subgroup.

Thus, in a preferred embodiment, the method of the invention comprises the step of identifying one or more subsets of plant cells comprising regenerated plant cells each having one or more highest concentrations of the desired nucleic acid sequence.

Thus, in one embodiment, as described herein, in analyzing the concentration of a desired nucleic acid sequence in the sample, the methods of the invention include the steps of extracting nucleic acid molecules (e.g., DNA and/or RNA) from the sample of each subset of genetically modified cells and disrupting the cells of the sample. For example, the cell sample taken for analysis in step (a) preferably comprises a minimum number of cells that gives an accurate prediction of the cell population in the subgroup. In one embodiment, DNA extraction is performed using about 30% or 15% or less cells of the subset, preferably less than, for example, 12%, 10%, 7%, 5%, 3%, 1%, 0,5%, 0,1% or less cells. The number of cells collected and analyzed in a subset of samples depends on the expected number of cells carrying the desired nucleic acid sequence.

For example, the efficiency of a genetic modification method (e.g., particle bombardment or agrobacterium-mediated transformation) can determine the abundance of a desired nucleic acid sequence in a population. Thus, the method for determining the concentration of the presence of a desired nucleic acid sequence in the DNA of a test cell is sensitive enough to detect the frequency of events.

As described herein, the concentration of a desired nucleic acid sequence in a sample may be determined by PCR-based analysis or nucleic acid sequencing of the relevant region (e.g., by qPCR, digital PCR, preferably digital micro-droplet PCR (ddPCR), or by high throughput sequencing methods, such as Next Generation Sequencing (NGS)). In a preferred embodiment, the concentration of the presence of the desired nucleic acid sequence at the target position in the genetically modified regenerative cells is determined by ddPCR. ddPCR was found to be well suited for determining the frequency in a fast and reliable manner. ddPCR may be used, for example, as described herein or as described, for example, in the following documents: WO 2018001884; or Peng C et al (2020), front.plant Sci. [ plant science front ] 11:610790; or Miyaoka Y et al, (2018) Methods in Molecular Biology (Clifton, n.j.) [ methods of molecular biology (klifton, n.jersey) ],1768,349-362.

Thus, in a preferred embodiment, the method of the invention comprises in a first step (a) genetically modifying (e.g. mutating, transforming or transfecting) plant cells comprising regenerated plant cells as described herein, dividing said modified cells into subgroups and then determining the concentration of the desired nucleic acid sequence in each subgroup, preferably by molecular screening (e.g. NGS or ddPCR).

In another embodiment, the methods of the invention comprise first dividing plant cells comprising regenerated plant cells into subgroups, then modifying (e.g., mutating, transforming or transfecting) cells in the subgroups, and preferably identifying the subgroup with the highest concentration of desired nucleic acid sequences by molecular screening (e.g., NGS or ddPCR). The concentration of the desired nucleic acid sequence in a representative sample of DNA for each subgroup is determined.

Thus, cell proliferation before or after genetic modification of the regenerative cells is not necessary.

According to the methods of the invention, the concentration of the desired nucleic acid sequence present is related to the relative number of genomes and thus to the relative number of cells carrying the desired nucleic acid sequence compared to the number of cells not carrying the desired nucleic acid sequence.

According to the methods of the invention, identifying one or more subsets having one or more highest concentrations, respectively, of the desired nucleic acid sequence concentration comprises determining one or more samples of each subset. For example, according to the invention, DNA is extracted from the cells to be tested and the concentration of the desired nucleic acid sequence is analyzed so that one or more subgroups with one or more highest concentrations can be identified.

According to the method of the invention, selecting one or more subgroups comprising plant cells comprising regenerative cells, each having one or more highest concentrations of the desired nucleic acid sequences, means selecting the appropriate number of subgroups for the next step from all subgroups tested.

The preferred selected number of subgroups depends on, for example, the number of cells in each subgroup, or the number of subgroups and/or the expected or predicted concentration of the desired nucleic acid sequence in the population. For example, if a lower number of cells carrying the desired nucleic acid sequence is found, the skilled artisan can select more subgroups than that number.

The size of the subgroups (e.g., the number of cells per subgroup) is selected by the skilled artisan in an appropriate manner. The preferred number of cells per subgroup depends on, for example, the total number of cells in the population, the efficiency of the modification process, and/or the concentration of the desired nucleic acid sequence in the population expected, predicted or predicted from the sample analyzed.

Preferably, if the selected subset has a high concentration of the desired nucleic acid sequence (e.g., above the average of all test samples in the same step), the subset is selected for the next step of the method of the invention. Preferably, the concentration of nucleic acid sequences in the selected subgroup is the highest concentration found in all subgroups.

In one embodiment, five, four, three, preferably two or one of the subgroups of the test subgroups having the highest concentration of the desired nucleic acid sequences are selected for the next step. A small number of selected subgroups is preferred, e.g. only one or two subgroups are selected in one step.

In one embodiment, for example, to produce a genetically modified plant, the modification efficiency is first determined and the genetically modified cell population comprising regenerated plant cells is divided into subgroups. In one embodiment, the efficiency of the genetic modification is not tested and the population is divided before or after the genetic modification process is performed.

Thus, in one embodiment, the method of the present invention comprises step (a) comprising, preferably consisting of:

a) (i) providing a population of plant cells comprising regenerated plant cells expected to have the desired nucleic acid sequence, (ii) dividing the population of plant cells into subgroups, (iii) extracting DNA and/or RNA from a sample of the cells from each subgroup, (iv) identifying one or more subgroups of the cells having the highest concentration of the desired nucleic acid sequence, and (v) selecting one or more subgroups of the cells having the highest concentration of the desired nucleic acid sequence,

Wherein, optionally, and in another embodiment, preferably, the cell is a plant cell comprising a genetically modified regenerated plant cell.

Thus, in another embodiment, the method of the present invention comprises step (a) comprising, preferably consisting of:

a) (i) dividing a population of plant cells comprising regenerated plant cells into subgroups, (ii) optionally, genetically modifying each subgroup of plant cells comprising regenerated plant cells, (iii) extracting DNA and/or RNA from a sample of said plant cells comprising regenerated plant cells of each subgroup, (iv) identifying one or more subgroups having the highest concentration of the desired nucleic acid sequences, and (v) selecting one or more subgroups of said cells having the highest concentration of the desired nucleic acid sequences,

Wherein, optionally, and in another embodiment, preferably, the plant cell comprising the regenerative plant cell is a plant cell comprising a genetically modified regenerative cell.

In one embodiment, the plant cell is of monocotyledonous or dicotyledonous origin. In one embodiment, the regenerated plant cells are of monocot or dicot origin.

In one embodiment, the population of cells (e.g., monocot or dicot cells) is derived from one or more independent genetic modification events, such as particle bombardment (e.g., particle gun shooting) or protoplast transfection batches. Each modification event (e.g., each transformation or transfection event) may be used as a subset. For example, cells from one shot construct a subset. In one embodiment, the population of regenerated plant cells is monocot or dicot, and the regenerated plant cells are derived from one or more independent genetic modification events, such as particle bombardment (e.g., particle gun shooting) or protoplast transfection batches.

a) (i) providing a population of plant cells comprising regenerated plant cells, (ii) dividing the population of plant cells comprising regenerated plant cells into subgroups and treating the cells of each subgroup with a genetic modification method (e.g. particle bombardment, agrobacterium transformation or protoplast transfection), (iii) extracting DNA and/or RNA from a sample of said cells from each subgroup, (iv) identifying one or more subgroups with the highest concentration of the desired nucleic acid sequences, and (v) selecting one or more subgroups of said cells with the highest concentration of the desired nucleic acid sequences.

In another embodiment, the population of cells treated by the genetic modification method (e.g., produced by particle bombardment or Agrobacterium transformation or protoplast transfection) is divided into different subgroups. For example, units of one shot are divided into several subgroups, or cells of several shots are mixed and then they are divided into subgroups.

In one embodiment of the invention, the concentration of the desired nucleic acid sequence is determined by: a sample of a subset of cells is obtained, the nucleic acid molecules of these cells are extracted, and the concentration of the desired nucleic acid sequence in the extract is determined. Extraction of DNA or RNA from cells produces non-renewable materials, such as the need to destroy cells.

In one embodiment, the method of the invention comprises selecting a sample from said plant cells comprising regenerated plant cells, e.g. 0.1%, 0,5%, 1,0%, 5,0%, 10%, 15%, 20%, 30%, 40% or 50% or more, if appropriate. Preferably, the selected sample is as small as possible, but large enough to quantitatively determine the concentration of the nucleic acid sequence. The size may for example depend on the efficiency of the genetic modification step, e.g. between 1% and 15% of cells per subgroup. In one embodiment, the selected sample comprises cells comprising regenerated plant cells.

In one embodiment, if it is desired to determine the concentration of the desired nucleic acid sequence in a cell sample representing cells particularly suitable for plant regeneration, the selected sample comprises only or predominantly regenerated plant cells. In one embodiment, if it is desired to select samples that avoid those regenerable cytopenias, for example, if the non-regenerable cells represent the concentration of a desired nucleic acid molecule in the non-regenerable cells, the selected sample comprises only or predominantly cells that are non-regenerable plant cells.

In the methods of the invention, substantially all cell types capable of dividing and proliferating may be used. In general, all regenerative cells can be used in the methods of the invention, whether regeneration occurs via organogenesis (e.g., budding) or by somatic/gametocyte embryogenesis. The invention may be adapted accordingly. The regenerative cells used in the method of the invention are plant cells.

The regenerated plant cells are for example selected from the group consisting of:

single cells, such as protoplasts or microspores,

Cell aggregates, such as cell suspensions or callus cultures,

Thus, preferably, the regenerated plant cells are selected from the group consisting of: protoplasts, microspores, cell suspensions, callus cultures, immature embryos, scutellum, cotyledons, roots, leaves, leaf bases, petioles, stems, meristems, roots, leaves, leaf bases, petioles, stems and meristems.

The regenerative cells used in the method of the present invention are not limited to these examples.

Thus, the method of the invention further comprises step (b) of culturing the regenerative cells of the subset selected in step (a) and producing, for example, callus or regenerated shoots.

In accordance with the present invention, in one embodiment, plant regeneration is initiated to produce a plant. Plant regeneration may begin with genetically modified (e.g., transformed or transfected) regenerative cells. It may also begin with unmodified (e.g., untransformed) tissue to select for cells that already exist in the plant with the desired trait.

"Recovery" or "regeneration" as used herein means regenerating plant cells by in vitro culture, such as plants produced from protoplasts or calli. It may be achieved, for example, via somatic embryogenesis or organogenesis. Methods for regenerating plants are well known in the art, e.g., haberlandt, g. (1902), sitzungsber aka. Wiss. Wien. Math. Nat. [ conference report of empire sciences, math-natural science ]111,69-91; skoog and Miller (1957), symp.Soc.exp.biol. [ society of laboratory Biol.Proprietary discussion ]54,118-130; steward et al (1958), am.J.Bot. [ J.America plant journal ]45,705-708. Key components of the development of media for in vitro plant regeneration are basal media, carbon sources, plant growth regulators and other added supplements to improve the regeneration step. The explant enters an induction period during which cells are identified to produce, for example, callus, shoots, roots or embryos. Explants or derived calli enter the stage of realization, which results in the emergence of shoots, roots and embryos, allowing the plants to be recovered and grown. In order to increase the efficiency of plant cell regeneration after transformation or genome editing, expression of genes encoding proteins involved in plant growth regulation, in particular so-called morphogenic genes, has proven useful (Gordon Kamm et al, plants [ plant ]2019,8,38; doi:10.3390/Plants 8020038). Transcription factors like WUSCHEL (WUS) and related WOX, BABY bottom or LEC1 are known to promote cellular development and somatic embryogenesis when overexpressed in monocots. This property has been used to increase the efficiency of genome editing in plants.

Typically, plant cells are transformed with a gene construct encoding a CRISPR-Cas complex and optionally with a gene construct encoding a morphogenic gene (WO 2018/224001, WO 2021/030242, WO 2021/022043). For example, ribonucleoprotein (RNP) is introduced, or a nucleic acid encoding a functional CRISPR-Cas system can be expressed (KANCHISWAMI PLANT CELL REP [ plant cell report ]2016 volume 35, 7 th edition, pages 1469-74; liang et al Nat Commun [ Nature. Communication ] 2017:14261).

Thus, the present invention includes increasing the efficiency of regeneration of genome-edited plant cells. For example, the methods of the invention include introducing into a plant cell, in the step of modifying the nucleic acid sequence of a regenerating cell, endonucleases and transcription factors (e.g., WUSCHEL (WUS), WOX, BABY bottom, LEC1, etc.) designed for targeted genomic modification in the form of proteins or their encoding mRNA, further comprising modifying the genome of the plant cell with the endonucleases and regenerating the plant cell into a plant, thereby allowing the transcription factors to increase the regeneration efficiency. In particular embodiments, the increased regeneration efficiency is an increased percentage of modified plant cells regenerated into mature plants as compared to a percentage of control plants regenerated from genome-edited plant cells, which control plants have undergone the same procedure except that they are not treated with a transcription factor (e.g., WUSCHEL (WUS), WOX, BABY bottom, or LEC 1).

In one embodiment, the cell population of the selected subset of cells is divided into secondary subsets prior to culturing the regenerating plant cells, e.g., prior to producing callus or shoots from the regenerating cells. The concentration of the desired nucleic acid sequences in the secondary subset is determined as described previously. Preferably, the concentration is determined by molecular screening (e.g., ddPCR or NGS). One or more secondary subsets having the highest concentration of the desired nucleic acid sequences are then selected and used to regenerate plants from the regenerated plant cells.

In one embodiment, after selecting regenerated plant cells from one or more selected subsets of step (a), i.e. before generating callus or shoots or plants from the regenerated cells from one or more selected subsets of step (a), the regenerated cells are divided into subsets and the presence of the nucleic acid sequence is determined as described before (preferably by molecular screening, e.g. ddPCR or NGS). One or more subsets of cells having the highest concentration of the desired nucleic acid sequence are then selected to produce the plant (step (c)).

A preferred method of determining the amount of nucleic acid sequence desired is ddPCR or NGS.

Thus, in one embodiment, the method of the invention comprises step (b) of obtaining a sample from plant cells comprising cultured cells (e.g., callus or shoots regenerated from cells of the selected subset of step (a)), extracting the DNA of the cells of the sample, and determining the amount of the desired nucleic acid molecule by ddPCR.

The concentration of the desired nucleic acid sequence in each sample comprising the subset of rejuvenated cells obtained in step (b) may be determined as described (e.g. according to the same principle as the rejuvenated cells of step (a)). For example, the concentration is determined after extracting nucleic acid molecules (e.g., DNA) from cells of the subset. The concentration of the desired nucleic acid sequence is preferably determined in PCR (particularly digital PCR, preferably ddPCR) or by sequencing the nucleic acid sequence (e.g., via NGS).

Thus, in one embodiment, the method of the present invention comprises step (b) comprising, preferably consisting of:

i) culturing one or more selected subsets of regenerative cells in step (a), (ii) extracting nucleic acid molecules (e.g., DNA) from a sample of the cultured cells (e.g., callus or regenerative shoots) of each subset, (iii) identifying one or more subsets having the highest concentration of desired nucleic acid sequences, and (iv) selecting one or more subsets of the cultured cells having the highest concentration of desired nucleic acid sequences.

Thus, in one embodiment, the method of the present invention comprises steps (a) and (b), comprising the steps of:

a) (a 1) (i) providing a population of plant cells comprising regenerated plant cells, (ii) dividing the population of plant cells comprising regenerated plant cells into subgroups, (iii) extracting nucleic acid molecules (e.g. DNA) from cells from one or more samples of said plant cells comprising regenerated cells from each subgroup, (iv) identifying one or more subgroups having the highest concentration of desired nucleic acid sequences, and (v) selecting one or more subgroups of said plant cells comprising regenerated plant cells having the highest concentration of desired nucleic acid sequences,

Wherein, optionally, the plant cell and/or regenerated plant cell is a genetically modified plant cell,

Or alternatively

(A2) (i) dividing a population of plant cells comprising regenerated plant cells into subgroups, (ii) optionally genetically modifying cells of a subgroup of plant cells comprising regenerated plant cells, (iii) extracting nucleic acid molecules (e.g. DNA) from one or more samples of said cells from each subgroup, (iv) identifying one or more subgroups having the highest concentration of desired nucleic acid sequences, and (v) selecting one or more subgroups of said cells having the highest concentration of desired nucleic acid sequences, and

B) (i) culturing cells from one or more selected subsets of step (a) (e.g., recovering callus or shoots from regenerated cells), (ii) extracting nucleic acid molecules (e.g., DNA) from cells from one or more samples from each subset, (iii) identifying one or more subsets having the highest concentration of desired nucleic acid sequences, and (iv) selecting one or more subsets of the cells having the highest concentration of desired nucleic acid sequences,

Wherein, optionally, the regenerated plant cell is a genetically modified regenerated plant cell, e.g., a cell produced by particle bombardment or Agrobacterium transfection, e.g., a cell produced by a gene editing process.

Furthermore, the method of the invention allows for recovery of plants from one or more of the subgroups selected in step (b). Thus, in one embodiment, the method of the present invention comprises step (c) comprising, preferably consisting of: culturing individual plants from one or more selected subsets of regenerated plant cells (e.g., regenerated calli or shoots), recovering the individual plants and/or growing the individual plants.

According to the invention, nucleic acid molecules, such as DNA, are extracted from one or more samples taken from a subset. Nucleic acid molecules extracted from one or more samples derived from a subset, such as extracted DNA, may be pooled. Alternatively, all cells from a subset of samples may be pooled and then nucleic acid molecules, such as DNA, extracted from the pooled cells.

Thus, in one embodiment, one or more subsets having one or more highest concentrations of the desired nucleic acid sequences are identified, and the plant is regenerated using cells (e.g., regenerated callus or shoots) of the one or more subsets.

According to the invention, in one step, after the plant has been recovered in step (c), the presence of the desired nucleic acid sequence in the individual plant is determined by extracting nucleic acid molecules (e.g. DNA) from one or more samples from said plant. The presence may be determined according to well known methods. The presence of the desired nucleic acid sequence is preferably determined in molecular sieves (e.g., in PCR, preferably ddPCR) or by sequencing the nucleic acid sequence (e.g., via NGR).

In one embodiment, the method of the invention comprises an additional step (ba) between steps (b) and (c):

(i) recovering individual plants from cells (e.g., calli or shoots) from one or more selected subsets of step (b), (ii) dividing a population of plants into subsets, (ii) extracting DNA from one or more samples from the plants from each subset, (iii) combining samples from individual plants from one subset and identifying one or more subsets having the highest concentration of the desired nucleic acid sequences, and (iv) selecting one or more subsets of the plants having the highest concentration of the desired nucleic acid sequences.

In one embodiment, in a subsequent step (v), an individual plant is selected from the subgroup having the highest concentration of the desired nucleic acid sequence.

Thus, in one embodiment, the method according to the invention, step (c) is after step (ba) as described.

To determine the presence and concentration of a desired nucleic acid sequence in a plant sample (if desired), plant tissue is harvested and the nucleic acid molecules are extracted from these samples. Preferably, the sample is obtained in a manner that allows for quantitative analysis.

Thus, in one embodiment, the method of the present invention preferably comprises the steps of:

(a1) (i) providing a population of plant cells comprising regenerated plant cells expected to have the desired nucleic acid sequence, (ii) dividing the population of plant cells comprising regenerated plant cells into subgroups, (iii) extracting DNA from one or more samples of said cells from subgroups, (iv) identifying one or more subgroups having the highest concentration of one or more desired nucleic acid sequences, and (v) selecting one or more subgroups of said cells having the highest concentration of desired nucleic acid sequences,

Wherein, optionally, the cell is a genetically modified cell, preferably a genetically modified regenerated plant cell,

Or alternatively

(A2) (i) dividing a population of plant cells comprising regenerated plant cells into subgroups, (ii) optionally, genetically modifying a subgroup of plant cells comprising regenerated plant cells, (iii) extracting DNA from one or more samples of said cells from each subgroup, (iv) identifying one or more subgroups having one or more highest concentrations of the desired nucleic acid sequences, and (v) selecting one or more subgroups of said cells having one or more highest concentrations of the desired nucleic acid sequences,

And

(B) (i) culturing one or more selected subsets of regenerated plant cells of step (a 1) or (a 2), (ii) extracting DNA from one or more samples of cultured plant cells (e.g., regenerated callus or shoots) from said subsets, (iv) identifying one or more subsets having one or more highest concentrations of the desired nucleic acid sequences, and (v) selecting one or more subsets of said cells (e.g., regenerated callus or shoots) having one or more highest concentrations of the desired nucleic acid sequences,

And

(C1) (i) recovering individual plants or individual shoots from the regenerated cells (e.g., regenerated calli or shoots) from one or more selected subsets of step (b), (ii) dividing the plant or shoot population into subsets, (ii) extracting DNA and/or RNA from one or more samples from cells of each subset, (iii) identifying one or more subsets having the highest concentration of one or more desired nucleic acid sequences, and (iv) selecting and growing one or more subsets of said plants or shoots having the highest concentration of one or more desired nucleic acid sequences,

Or alternatively

(C2) (i) recovering individual plants or individual shoots from one or more selected subsets of regenerated cells (e.g., callus or regenerated shoots) from step (b), (ii) obtaining a sample comprising DNA and/or RNA from each plant or each shoot, (iii) analyzing the DNA and/or RNA for the presence of a desired nucleic acid sequence, (iv) selecting and growing plants having the desired nucleic acid sequence,

Wherein, optionally, the plant cell is a genetically modified cell, preferably a regenerated plant cell, e.g. a cell produced by particle bombardment or agrobacterium transformation or protoplast transfection.

In one embodiment, step (b) is omitted. For example, step (b) is optional if the efficiency of modification of the regenerative cells in step (a) is higher as described herein. For example, step (b) may be omitted after the effective bombardment.

As discussed, in one embodiment, the method further comprises a step (ba), such as step (a) (e.g., (a 1) or (a 2)), step (ba), and step (c) (e.g., (c 1) or (c 2)).

The cultivation in step (c) of the present invention may be carried out, for example, in a greenhouse or in a field.

Thus, in one embodiment of the invention, the regenerative cells are further developed into a single clonal plant.

Thus, in one embodiment, the method of the invention comprises step (a) (e.g., (a 1) or (a 2)) and step (c) (e.g., (c 1) or (c 2)) as described herein, and optionally step (b 1) or (b 2), e.g., steps (b 1) and (ba) or steps (b 2) and (ba), each in combination with step (a 1) or (a 2). Thus, in one embodiment, the method of the present invention consists of the steps of: step (a) (e.g., (a 1) or (a 2)), step (b) and step (c) (e.g., (c 1) or (c 2)), and optionally step (ba).

Thus, in one embodiment, the method of the present invention comprises the steps of:

-a1 and c1;

-a1 and c2;

-a2 and c1; or alternatively

-A2 and c2.

In a preferred embodiment, the method of the present invention comprises the steps of:

-a1 and c1;

-a1 and c2;

-a2 and c1; or alternatively

-A2 and c2,

In combination with the following steps:

b1 or b2.

For example, the method of the invention comprises, preferably consists of:

-a1、b1、c1；

-a2、b1、c1；

-a1、b2、c1；

-a1、b1、c2；

-a1、b2、c2，

-a2、b1、c2；

-a2, b2, c1; or alternatively

-a2、b2、c2；

Or any other combination of steps in the order (a) to (c). In one embodiment, each combination includes step (ba).

In one embodiment, the selected event is molecularly characterized, for example, in a sequence-specific analysis. These subsequent analyses will reveal, for example, allele-specific mutation profiles and types of modification on different subgenomic groups.

According to the present invention, "one or more samples" means that the person skilled in the art suitably selects a plurality of samples taking into account the number of individual members of the group and/or the efficiency of the modification step.

Thus, in one embodiment of the invention, the method for modifying regenerative cells comprises protein or DNA delivery, such as, for example, delivery by a gene gun. In one embodiment, the genome is edited, but no nucleic acid molecules are introduced into the genome of the target cell. Thus, in one embodiment, the invention includes the step of genetically modifying the cell using a CRISPR/Cas system (e.g., in base editing or lead editing).

In one embodiment, the regenerative cells used in the methods of the invention are plant cells or cells derived from plants (particularly monocotyledonous and dicotyledonous plants, including forage or forage legumes, ornamental plants, food crops, trees or shrubs). According to an embodiment of the invention, the plant is a crop plant.

Thus, in one embodiment, the regenerative cells are monocot regenerative cells. According to another embodiment of the invention, the plant produced is cereal or a derivative thereof. Preferred are monocotyledonous crop plants. According to another embodiment of the invention, the plant is sugarcane, and the cereal includes rice, maize, wheat, barley, millet, rye, triticale, sorghum, secale, spelt, monococcus, bran, milo (milo) and oats. In certain embodiments, the one or more plants of the invention or used in the methods of the invention are selected from the group consisting of: corn, wheat, rice, soybean, cotton, canola (including canola), sugar cane, sugar beet and alfalfa.

RNP-mediated genome editing and plant regeneration has been shown to be successful for several monocots using particle bombardment, such as rice (Banakar et al 2020, rice [ Rice ] 13:4.), maize (SVITASHEV et al 2016, nat. Commun [ Nature. Communication ] 7:13274), and wheat (Liang et al 2017, nat. Commun [ Nature. Communication ] 8:14261).

For example, the monocot She Zaisheng cell is for example selected from the group consisting of:

single cells, such as protoplasts or microspores,

Cell aggregates, such as cell suspensions or callus cultures,

Thus, preferably, the monocotyledonous regenerating plant cell is selected from the group consisting of: protoplasts, microspores, cell suspensions, callus cultures, immature embryos, scutellum, cotyledons, roots, leaves, leaf bases, petioles, stems, meristems, roots, leaves, leaf bases, petioles, stems and meristems.

In one embodiment, the monocot She Zai producer cell is a genetically modified monocot producer cell, e.g., a transformed and/or transfected monocot producer cell. In one embodiment, the mutated monocot regenerative cells are cells that are bombarded cells in a gene gun delivery system.

In a preferred embodiment, the cells used in the method of the invention are wheat cells or are derived from wheat cells.

In another embodiment, the regenerative cells are dicotyledonous regenerative cells.

Effective methods for generating genome-edited dicotyledonous plants are well known to those skilled in the art, using CRISPR DNA components delivered in the following manner: agrobacterium (as for example for canola (Braatz et al 2017,Plant Physiol [ plant physiology ]. 6 months; 174 (2): 935-942)), cotton (Li et al 2017, sci Rep [ science report ]3 months 3 days; 7:43902 ]) or genomics (as for example for soybean (Li et al 2015,Plant Physiol [ plant physiology ]. 10 months; 169 (2): 960-970)), or protoplast transfection (as for example for potato (Anderson et al PLANT CELL Reports [ plant cell report ]. Volume 36, 117-128)), or CRISPR Ribonucleoprotein (RNP) edited as a DNA-free genome in protoplasts (as for example for lettuce (Woo et al 2015, nat. Biotechnol [ natural. Biotechnology ] 33:1162-1164), cabbage (Park et al 2019,Plant Biotechnol.Rep. [ plant biotechnology report ] 13:483-489) or (potato zan 2020,Front.Plant Sci, front edge science report [ 10:1649)).

Dicotyledonous crop plants are preferred. Examples of crop plants include, but are not limited to, chicory, carrot, cassava, clover, soybean, beet, sugar beet, sunflower, canola, alfalfa, rapeseed, flaxseed, cotton, tomato, potato, tobacco, and Brassica (Brassica) species (e.g., cabbage (Brassica oleracea) or Brassica napus (Brassica napus) or mustard (Brassica juncea)).

Thus, the method of the invention thus comprises recovering cultured callus or shoots from said regenerated dicotyledonous cells in one or more subgroups having one or more highest concentrations of the desired nucleic acid sequences, and selecting one or more subgroups having the presence of one or more highest concentrations of the desired nucleic acid sequences. Thus, in one embodiment, the plant tissue cultivated in step (b) of the invention is a callus or bud.

In one embodiment, the dicotyledonous regenerating cell is a mutated dicotyledonous regenerating cell, e.g., a transformed or transfected dicotyledonous regenerating cell. The mutated dicotyledonous regenerative cells can be produced, for example, in a gene gun delivery system, or in transfection of protoplasts, microspores, etc., or in other methods known in the art or described below. Thus, in one embodiment, the mutated dicotyledonous regenerative cells are cells that are bombarded in a gene gun delivery system.

Thus, the present invention also relates to methods for producing dicotyledonous shoots having a desired nucleic acid sequence and growing dicotyledonous plants from the selected shoots.

The regenerated dicotyledonous cells used in the methods of the invention can be single cells (e.g., protoplasts, microspores), cell aggregates (cell suspensions and callus cultures), multicellular seedling explants (roots, hypocotyls, cotyledons, leaves and meristematic tissue), multicellular plant explants (leaves, stems, petioles), or mature seed explants (meristematic tissue, cotyledons).

Thus, in one embodiment, the method of the present invention thus comprises the steps of:

a)

introducing into a population of plant cells comprising regenerated plant cells (e.g., explants) a composition comprising a system capable of introducing mutations at predefined sites in the genome,

Selecting samples from said cells, e.g. 0.1%, 0,5%, 1,0%, 5,0%, 10%, 15%, 20%, 30%, 405, 50%, 60%, 70% and at most 80% (preferably as few as possible, depending on the efficiency of the genetic modification step, e.g. between 1% and 15%) of cells per plate,

-Extracting DNA from the sample cells, wherein, optionally, the respective cells are fully used for extraction and combining DNA from cells of one or more plates, or combining cells for DNA extraction, wherein, optionally, the respective cells taken are fully used for extraction, preferably thereby extracting DNA from the cell pool, such that a single culture dish or a single shot with a cell culture dish can be identified as source of sample, and

Analyzing the concentration of the desired sequence in the extracted DNA in a molecular sieve, for example using ddPCR or NGS, and determining the concentration of the mutation or sequence at a predefined position in the genome,

Selecting a plate based on the result of said analysis, for example preferably 1%, 5%, 10% or 25%, for further culturing,

And

b)

Growing callus, shoots, roots and/or plants in tissue culture from regenerative cells of selected plates, and

Selecting samples from said regenerated calli, shoots, roots and/or plants in tissue culture, e.g. 0.1%, 0,5%, 1,0%, 5,0%, 10%, 15%, 20%, 30%, 405, 50%, 60%, 70% and up to 80% (preferably as few as possible, depending on the efficiency of the genetic modification step, e.g. between 1% and 15%) of plant material, e.g. regenerated calli, shoots, roots and/or plants in tissue culture,

Extracting DNA from cells in said sample of regenerated callus, shoots, roots and/or plants in tissue culture, wherein cells in the sample of regenerated callus, shoots, roots and/or plants in tissue culture are used entirely for extraction and DNA from cells of one or more cassettes are pooled or pooled cells are used for DNA extraction, preferably so that DNA is extracted from said cell pool, so that a single culture dish or single shot with a cell culture dish can be identified as the source of the sample, and

Analyzing the extracted DNA for the presence of a desired mutation or sequence at a predefined position in the genome using ddPCR or NGS and determining the concentration of the mutation or sequence at the predefined position in the genome,

Selecting a cassette with the best or highest concentration of mutation or desired sequence, e.g.preferably 1%, 5%, 10% or 25%, based on the results of the analysis, for further cultivation,

And

c)

Further culturing and regenerating regenerated calli, shoots, roots and/or plants in tissue culture into plants, for example further developing monoclonal plants,

Selecting samples from plants (e.g. from leaves), preferably leaf samples of the same size, e.g. leaf discs,

-Extracting DNA from a plant sample,

-Analysing the DNA and determining the concentration of the desired mutation or sequence at the predefined position, and

Selecting plants comprising the mutation or the desired sequence,

Wherein step (b) is optional. In a preferred embodiment, the method of the present invention comprises steps (a), (b) and (c).

In one embodiment, the invention includes selecting plants that also exhibit preferred phenotypic or agronomic characteristics (e.g., increased yield, stress tolerance, etc.) in step (c). Thus, step (c) may for example be used to determine a single genotype (clone characterization) of a particular sample (e.g. a plant sample). Thus, in one embodiment, the samples obtained in step (c) are not combined and each sample is analyzed separately, e.g., if the plant is grown in a greenhouse

Thus, according to the method of the present invention, steps (a) and (b) are used to reduce the number of plants to be screened in step (c), thereby reducing costs and increasing yield.

Furthermore, in one embodiment, in the process of the present invention comprising steps (a) to (c) as described herein, the product of step (a) is directly used in step (b) without any further treatment of the product of step (a) prior to step (b).

In another embodiment, the product of step (b) of the process of the invention is used directly in step (c) of the process of the invention without any further treatment of the product before it is used in step (c). In one embodiment, in the process of the present invention, the product of step (a) is used directly in step (b), and the product of step (b) is used directly in step (c) of the process. Thus, in one embodiment, the products of step (a) and step (b) are not modified. Thus, in one embodiment, the method of the present invention consists of steps (a) to (c).

Examples of plants in which cells may be used in the methods of the invention include maple species (Acer spp.), kiwi species (ACTINIDIA spp.), okra species (Abelmoschus spp.), sisal (AGAVE SISALANA), agropyron species (Agropyron spp.), creeping bentgrass (Agrostis stolonifera), allium species (Allium spp.), amaranthus species (Amaranthus spp), european seashore grass (Ammophila arenaria), Pineapple (Ananas comosus), annona species (Annona spp.), celery (Apium graveolens), arachis species (ARACHIS SPP), jackfruit species (Artocarpus spp.), asparagus (Asparagus officinalis), avena species (Avena spp.) (e.g., oat (AVENA SATIVA), wild oat (Avena fatua), red oat (Avena byzantina), Wild oat variety sativa (Avena fatua var. Sativa), hybrid oat (Avena hybrid)), carambola (Averrhoa carambola), trifoliate acanthopanax species (Bambusa sp.), white gourd (Benincasa hispida), brazil chestnut (Bertholletia excelsea), beet (Beta vulgaris), brassica species (Brassica spp.) (e.g., brassica napus, guan subspecies (Brassica rapa ssp.) [ canola ], rape, brassica napus (turnip rape) ]), kadaba (Cadaba farinosa), tea (CAMELLIA SINENSIS), canna (CANNA INDICA), canna (Cannabis sativa), capsicum species (Capsicum spp.), sedge (Carex elata), papaya (CARICA PAPAYA), pseudostellaria macrophylla (Carissa macrocarpa), hickory species (Carya spp.), cannabis sativa, Safflower (Carthamus tinctorius), chestnut species (Castanea spp.), gecko (Ceiba pentandra), chicory (Cichorium endivia), camphorwood species (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus species (Citrus spp.), coconut species (Cocos spp.), coffee species (coffire spp.), taro (Colocasia esculenta), taro (v/v), Cola species (Cola spp.), jute species (Corchorus sp.), coriander (Coriandrum sativum), hazelnut species (Corylus spp.), crataegus species (Crataegus spp.), saffron (Crocus sativus), cushaw species (Cucurbita spp.), cucumber species (cusumis spp.), cynara species (Cynara spp.), carrot (Daucus carota), Beggarweed species (Desmodium spp.), longan (Dimocarpus longan), dioscorea species (Dioscorea spp.), diospyros species (Diospyros spp.), barnyard species (Echinochloa spp.), oil palm (Elaeis) (e.g., oil palm (Elaeis guineensis), oil palm americana (Elaeis oleifera)), Eleusine seed (Eleusine coracana), meadowrus frequency (Eragrostis tef), a seed of Manchurian euphorbia herb (America seed of Manchurian euphorbia herb, a seed of Manchurian euphorbia herb (Amarum seed of Manchurian euphorbia herb), The species of the genus festuca (Erianthus sp.), loquat (Eriobotrya japonica), eucalyptus (Eucalyptus sp.), red fruit (Eugenia uniflora), buckwheat (Fagopyrum spp.), cyclobalanopsis (Fagus spp.), festuca (Festuca arundinacea), fig (Ficus carica), kumquat (Fortunella spp.), strawberry (Fragaria spp.), and the like, Gingko (Ginkgo biloba), glycine spp (e.g., soybean (Glycine max), soybean (Soja hispida), or soybean (Soja max)), cotton upland (Gossypium hirsutum), sunflower spp (e.g., sunflower (Helianthus annuus)), hemerocallis (Hemerocallis fulva), hibiscus spp, and combinations thereof Barley species (Hordeum spp.) (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), juglans species (Juglans spp.)), lettuce (Lactuca sativa), mucuna species (Lathyrus spp.), lentils (Lens collinearis), flax (Linum usitatissimum), litchi (LITCHI CHINENSIS), vensis species (Lotus spp.), The plant species may include, for example, lupinus (Luffa acutangula), lupinus spp, red bayberry (Luzula sylvatica), lycopersicon (Lycopersicon spp), such as tomato (Lycopersicon esculentum), cherry tomato (Lycopersicon lycopersicum), pear tomato (Lycopersicon pyriforme), sclerotium (Macrotyloma spp), and the plant species may include, for example, lupinus (scopinus spp), Malus species (Malus spp.), acerola (MALPIGHIA EMARGINATA), malus pumila (MAMMEA AMERICANA), mango (MANGIFERA INDICA), tapioca species (Manihot spp.), pistachio (MANILKARA ZAPOTA), alfalfa (Medicago sativa), sweet clover species (Melilotus spp.), mentha species (Mentha spp.), mango (Miscanthus sinensis), Balsam pear species (Momordica spp.), black mulberry (Morus nigra), musa species (Musa spp.), nicotiana species (Nicotiana spp.), olea species (Olea spp.), opuntia species (Opuntia spp.), butyrospermum species (Ornithopus spp.), oryza species (Oryza spp.) (e.g., rice (Oryza sativa), broadleaf rice (Oryza latifolia)), millet (Panicummiliaceum), oryza sativa, Switchgrass (Panicum virgatum), passion flower (Passiflora edulis), parsnip (PASTINACA SATIVA), pennisetum species (Pennisetum sp.), avocado species (Persea spp.), parsley (Petroselinum crispum), phalaris (Phalaris arundinacea), phaseolus species (Phaseolus spp.), timothy grass (Phleum pratense), timothy grass (spp.), and combinations thereof, The plant species may include, for example, a horseradish species (Phoenix spp.), a reed (PHRAGMITES AUSTRALIS), a Physalis spp.), a Pinus species (Pinus spp.), pistachio PISTACIA VERA, a Pisum spp.), a poacha species (Poa spp.), a Populus spp, a mesquite species (Populus spp.), a mesquite species (Prosopis spp.), a prune species (Prunus spp), a guava species (Psidium spp), a Psidium spp, Pomegranate (Punica granatum), pear (pyris communis), oak species (Quercus spp.), radish (Raphanus sativus), rheum officinale (Rheum rhabarbarum), black currant species (Ribes spp.), castor bean (Ricinus communis), rubus species (Rubus spp.), saccharum species (Saccharum spp.), salix species (Salix sp.), and black currant species (Ribes spp.), hemp (Ricinus communis), rubus species (Rubus spp.), saccharum species (Saccharum spp.), salix species (Salix sp.), Sambucus species (Sambucus spp.), rye (SECALE CEREALE), sesbania species (Sesamum spp.), sinapis species (Sinapis sp.), solanum species (Solanum spp.) (e.g., potato (Solanum tuberosum), red eggplant (Solanum integrifolium), or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach species (Spinacia spp.), the species of genus syzygium (Syzygium spp.), the species of marigold (Tagetes spp.), the species of taraxacum (Tamarindus indica), the species of cocoa (Theobroma cacao), the species of axletree (Trifolium spp.), the species of festuca arundinacea (Tripsacum dactyloides), the species of triticale (Triticosecale rimpaui), the species of wheat (Triticum spp), for example, wheat (Triticum aestivum), Durum wheat (Triticum durum), cone wheat (Triticum turgidum), wheat (Triticum hybernum), mojia wheat (Triticum macha), float wheat (Triticum sativum), one-grain wheat (Triticum monococcum) or common wheat (Triticum vulgare)), trollius chinensis (Tropaeolum minus), trollius chinensis (Tropaeolum majus), mojia wheat (Triticum aestivum), Vaccinium species (Vaccinium spp.), vicia spp), vicia species (Vigna spp), vigna species (Vigna spp), herba Violae (Viola odorata), vitis species (Vitis spp), semen Maydis (Zea mays), oriental water cherry (Zizania palustris), zizyphus species (Ziziphus spp), etc.

The invention also provides plants produced by the methods of the invention. Such plants may have an improved phenotype or agronomic trait, for example due to genetic modification of the regenerating cells in step (a) of the method of the invention. A trait of particular economic interest is increased yield. Yield is generally defined as the measurable economic value yield of a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, such as the number and size of organs, plant type (e.g., number of branches), seed production, leaf senescence, etc. Root development, nutrient uptake, stress tolerance and early vigor may also be important factors in determining yield. Thus, optimizing the above-mentioned factors may help to increase crop yield. Another important trait for many crops is early vigour. Improving early vigor is an important goal of modern rice breeding programs in both temperate and tropical rice varieties. Long roots are important for proper soil anchoring of hydroponic rice. Longer shoots are associated with vigor in the case of direct sowing of rice into flooded fields, and in the case of plants which must emerge from water rapidly. In the case of drill seeding, longer mesocotyls and coleoptiles are important for good emergence. The ability to provide early vigor to plants would be very important in agriculture. Another important trait is improved abiotic stress tolerance. Abiotic stress is a major cause of crop losses worldwide, reducing the average yield of most major crop plants by more than 50% (Wang et al, planta [ botanic ]218,1-14,2003). Abiotic stress may be caused by drought, salinity, extreme temperatures, chemical toxicity and oxidative stress. The ability to increase the tolerance of plants to abiotic stress would have great economic advantages to farmers worldwide and would allow crops to be planted under adverse conditions and in areas where they would otherwise not be able to. Another important trait is resistance to biotic stress, typically caused by pathogens (such as bacteria, viruses, fungi, plants, nematodes and insects, or other animals that may have a negative impact on plant growth).

Plants produced by the methods of the invention may also have improved nutritional traits, such as higher levels of protein, minerals, vitamins, micronutrients, essential amino acids, or other health promoting compounds.

The invention further extends to encompass the progeny of a cell, tissue, organ or whole plant that has been produced by any of the above-mentioned methods, the only requirement being that the progeny exhibit the same characteristics (i.e., at least the same genomic modification and/or the same improved agronomic trait) as the parent plant, however, provided that the plant is produced by the methods of the invention and not by a process that is biological in nature, and provided that the progeny is different from and distinguishable from a naturally occurring plant.

The invention also extends to harvestable parts of a plant produced by any of the above mentioned methods, such as but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs, which harvestable parts comprise the desired editing or modification in the genome.

Furthermore, the present invention relates to products derived or produced (preferably directly derived or produced) from harvestable parts of such plants, such as dry granules, pressed stems, coarse powders or powders, oils, fats and fatty acids, carbohydrates, juices, liquids or proteins. Preferred carbohydrates are starch, cellulose or sugar (preferably sucrose). Also preferred products are residual dry fibers, molasses or filter cakes (e.g. from sugar cane), such as stems (like bagasse from sugar cane after removal of the juice). In one embodiment, the product still contains a genome or portion thereof that contains the desired edits or modifications that can be used, for example, as an indicator of the particular quality of the product. In another embodiment, the product derived or produced from a plant according to the invention is different from the product derived or produced from a plant that does not comprise genome editing.

The invention also includes methods for making a product comprising a) growing a plant of the invention, and b) producing the product from a plant of the invention or a part thereof (e.g., a stem, root, leaf, and/or seed) or from a plant of the invention or a part thereof. In further embodiments, the methods comprise the steps of: a) growing the plant of the invention, b) removing the harvestable parts as described herein from the plant, and c) producing said product from or with the harvestable parts of the plant according to the invention.

In further embodiments, the product produced by the manufacturing method of the present invention is a plant product such as, but not limited to, a food, feed, food supplement, feed supplement, fiber, cosmetic, or pharmaceutical. In another embodiment, the agricultural products are prepared using a production method such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.

In yet another embodiment, the nucleic acid comprising the desired edits or modifications produced using the methods of the invention is contained in an agricultural product. In particular embodiments, the nucleic acids of the invention comprising the desired edits or modifications may be used as product markers, for example in the case of agricultural products produced by the methods of the invention. Such a marking can be used to identify products that have been produced by an advantageous method, which not only results in a higher efficiency of the method, but also improves the quality of the product due to the improved quality of the plant material and harvestable parts used in the method. Such labels may be detected by a variety of methods known in the art, such as, but not limited to, PCR-based methods for nucleic acid detection or antibody-based methods for protein detection.

The invention also encompasses the use of nucleic acids comprising a desired edit or modification, which may be used in breeding programs wherein DNA markers are identified that may be genetically related to nucleic acids or loci on a genome comprising the desired edit or modification. Nucleic acids themselves containing the desired edits or modifications can be used to define molecular markers. This DNA marker can then be used in a breeding program to select plants having one or more enhanced yield-related traits as defined herein in the methods of the invention. Nucleic acids comprising the desired edits or modifications can also be used as probes for genetic and physical mapping of genes of which they are a part, and as markers for traits related to those genes. Such information can be used in plant breeding to develop lines with a desired phenotype.

The invention also provides a platform for producing a genome-edited plant, the platform comprising means for selecting a target gene for editing and designing a suitable gRNA that allows for a desired modification of the target gene; a second module for performing gene editing, comprising preparing the cells to be edited, introducing an endonuclease designed for the desired genomic modification, for example together with TaWOX, in the form of a protein or mRNA according to the method of the invention; a third module for regenerating the edited cells into plants; and a screening system for selecting plants having a desired genome editing. The genome-edited plants produced with this platform can then be used in a breeding scheme.

The invention also encompasses sowing seeds from plants produced by the invention and growing a population of offspring. For example, seeds are grown in a greenhouse or field. Samples from the offspring produced, such as samples from leaves or seeds, may be analyzed, for example, by NGS. Preferably, the plant is selected from the progeny comprising the predicted mutation and isolated as desired.

Detailed Description

The method of introducing the desired genomic or genetic modification (as protein or as mRNA) may be accomplished chemically, non-chemically or by physical means. Chemical means for introducing proteins or nucleic acids in cells are generally dependent on uptake by endocytosis or on incorporation into the cell membrane. Non-limiting examples of chemical means for introduction include liposome transfection, polyethyleneimine (PEI) mediated introduction, polyethylene glycol (PEG) mediated introduction, nuclear transfection, calcium phosphate precipitation, liposomes, immunoliposomes, fusion, polycations or lipids: nucleic acid conjugates, cell penetrating peptides and DEAE-dextran mediated transfection. Non-chemical means encompass methods that create pores in the cell membrane or points of increased membrane permeability. Well known examples are electroporation, sonoporation (cavitation by bubbles) and the use of lasers. Non-limiting examples of physical means for introduction include microinjection, nanoparticle-mediated delivery, particle gun technology (gene gun method), and puncture transfection (through the use of needle-like nanostructures coated with one or more compounds of interest). For example, the desired genomic modification may be introduced by physical means, preferably by gene gun. Gene gun or particle bombardment is a tool for delivering one or more compounds into cells by coating the compounds on metal microparticles (typically tungsten or gold particles) and firing these coated particles into the cells with a gene gun. This technique has proven to be very useful for transforming plants that are otherwise difficult to transform or regenerate, as well as for transforming organelles such as protoplasts (for reviews, see Ozyigit and Kurtoglu, mol Biol Rep [ report on molecular biology ]47 (12): 9831-9847, 2020). Another useful method is aerosol beam microinjection (US 5,240,842).

Abbreviations: GFP-green fluorescent protein, GUS-beta-glucuronidase, BAP-6-benzylaminopurine; 2,4-D-2, 4-dichlorophenoxyacetic acid; MS-Murrager and Scott medium; NAA-1-naphthylacetic acid; MES,2- (N-morpholino-ethanesulfonic acid, IAA indoleacetic acid, kan, kanamycin sulfate, GA 3-gibberellic acid, timesin ^TM, ticarcillin disodium/clavulanate potassium, microl.

It is to be understood that the invention is not limited to the particular methodology or scheme. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a carrier" is a reference to one or more carriers and includes equivalents thereof known to those skilled in the art, and so forth. The term "about" is used herein to mean about, approximately, or around … …. When the term "about" is used in connection with a range of values, it modifies that range by extending the boundaries above and below the numerical values set forth. Generally, the term "about" is used herein to modify a numerical value above and below that stated value with a 20% (preferably 10%) difference, either upward or downward (higher or lower). As used herein, the word "or" means any one member of a particular list and also includes any combination of members of the list. When used in this specification and the following claims, the terms "comprises," "comprising," "includes," "including," "includes" and "including" are intended to specify the presence of one or more stated features, integers, components or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps or groups thereof. For clarity, certain terms used in this specification are defined and used as follows:

Donor DNA molecule: as used herein, the terms "donor DNA molecule," "repair DNA molecule," or "template DNA molecule," as used interchangeably herein, mean a DNA molecule having a sequence to be introduced into the genome of a cell. It may flank sequences homologous or identical to sequences in a target region of the genome of the cell at the 5 'and/or 3' end. It may comprise sequences which are not naturally occurring in the corresponding cell, such as ORFs, non-coding RNAs or regulatory elements which should be introduced into the target region, or it may comprise sequences which are homologous to the target region, except for at least one mutation, gene editing: the sequence of the donor DNA molecule may be added to the genome or it may replace a sequence in the genome that is the length of the donor DNA sequence.

Double-stranded RNA: a "double stranded RNA" molecule or "dsRNA" molecule comprises a sense RNA fragment of a nucleotide sequence and an antisense RNA fragment of a nucleotide sequence, both of which comprise nucleotide sequences that are complementary to each other, allowing the sense and antisense RNA fragments to pair and form a double stranded RNA molecule.

Endogenous: an "endogenous" nucleotide sequence refers to a nucleotide sequence that is present in the genome of an untransformed plant cell.

Enhanced expression: "enhancing" or "increasing" the expression of a nucleic acid molecule in a plant cell is used herein equally well and means that the expression level of the nucleic acid molecule in a plant, plant part or plant cell after application of the method of the invention is higher than the expression level of the nucleic acid molecule in a plant, plant part or plant cell before application of the method or is higher than the expression level in a reference plant lacking the recombinant nucleic acid molecule of the invention. For example, the reference plant comprises the same construct lacking only the corresponding NEENA. The term "enhanced" or "increased" as used herein is synonymous and means herein a higher, preferably significantly higher, expression of a nucleic acid molecule to be expressed. As used herein, "enhancing" or "increasing" the level of a substance (agent) (e.g., protein, mRNA, or RNA) means increasing the level relative to a substantially identical plant, portion of a plant, or plant cell lacking a recombinant nucleic acid molecule of the invention (e.g., lacking a NEENA molecule, recombinant construct, or recombinant vector of the invention) grown under substantially identical conditions. As used herein, a "increase" or "increase" in the level of a substance (e.g., preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by a target gene and/or a protein product encoded thereby) means an increase in the level by 50% or more, e.g., 100% or more, preferably 200% or more, more preferably 5% or more, even more preferably 10% or more, most preferably 20% or more, e.g., 50% relative to a cell or organism lacking the recombinant nucleic acid molecule of the invention. The enhancement or increase may be determined by methods familiar to the skilled artisan. Thus, an increase or increase in the amount of nucleic acid or protein may be determined, for example, by immunological detection of the protein. In addition, techniques such as protein assays, fluorescence, RNA hybridization, nuclease protection assays, reverse transcription (quantitative RT-PCR), ELISA (enzyme linked immunosorbent assay), western blotting, radioimmunoassay (RIA) or other immunoassays and fluorescence activated cell analysis (FACS) can be used to measure specific proteins or RNAs in plants or plant cells. Depending on the type of protein product induced, its activity or effect on the phenotype of the organism or cell may also be determined. methods for determining the amount of protein are known to the skilled artisan. Examples which may be mentioned are: micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest [ Scandina clinical and laboratory research journal ] 5:218-222), folin-Ciocalteau method (Lowry OH et al (1951) J Biol Chem [ journal of biochemistry ] 193:265-275) or measurement of CBB G-250 absorption (Bradford MM (1976) analytical Biochem [ analytical biochemistry ] 72:248-254). as one example of quantifying protein activity, the detection of luciferase activity is described in the examples below.

Expression: "expression" refers to biosynthesis of a gene product, preferably to transcription and/or translation of a nucleotide sequence (e.g., an endogenous gene or a heterologous gene) in a cell. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and optionally subsequent translation of the mRNA into one or more polypeptides. In other cases, expression may refer only to transcription of the DNA harboring the (harbouring) RNA molecule.

Expression construct: as used herein, "expression construct" means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate plant part or plant cell, comprising a promoter functional in said plant part or plant cell into which it is to be introduced, operably linked to a nucleotide sequence of interest, optionally operably linked to a termination signal. If translation is desired, it will typically also contain sequences required for proper translation of the nucleotide sequence. The coding region may encode a protein of interest, but may also encode a functional RNA of interest, such as RNAa, siRNA, snoRNA, snRNA, a microRNA, a ta-siRNA, or any other non-coding regulatory RNA, in sense or antisense orientation. Expression constructs comprising a nucleotide sequence of interest may be chimeric, meaning that one or more of its components is heterologous with respect to one or more of the other components. The expression construct may also be one that occurs naturally, but has been obtained in recombinant form for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e., the particular DNA sequence of the expression construct is not naturally present in the host cell and must have been introduced into the host cell or into the progenitor cell of the host cell by a transformation event. Expression of the nucleotide sequence in the expression construct may be under the control of a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of plants, the promoter may also be specific to a particular tissue, or organ, or stage of development.

Exogenous (foreign): the term "exogenous" refers to any nucleic acid molecule (e.g., a gene sequence) that is introduced into the genome of a cell by experimental manipulation, and may include sequences found in the cell, so long as the introduced sequence contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) and is therefore different relative to the naturally occurring sequence.

Functional connection: the term "functionally linked" or "functionally linked" is understood to mean, for example, that the regulatory element (e.g. promoter) is arranged in sequence with the nucleic acid sequence to be expressed and, if appropriate, with further regulatory elements (e.g. such as, for example, a terminator or NEENA) in the following manner: such that each of these regulatory elements is capable of performing its intended function to permit, modify, facilitate or otherwise affect expression of the nucleic acid sequence. As synonyms, the expression "operatively connected" or "operatively connected" may be used. The outcome of expression depends on the arrangement of the nucleic acid sequence relative to the sense or antisense RNA. For this purpose, a direct connection in the chemical sense is not necessarily required. Genetic control sequences (such as, for example, enhancer sequences) may also exert their function on the target sequence from a more distant location or a location that is actually remote from other DNA molecules. A preferred arrangement is one in which the nucleic acid sequence to be expressed recombinantly is located after the sequence acting as a promoter, such that the two sequences are covalently linked to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs. In a preferred embodiment, the nucleic acid sequence to be transcribed is located behind a promoter, so that the transcription initiation is identical to the desired initiation of the chimeric RNA of the invention. Functional ligation and expression constructs may be generated by means of conventional recombinant and cloning techniques as described, for example, in the following: MANIATIS T, FRITSCH EF and Sambrook J (1989) Molecular Cloning: ALaboratory Manual [ molecular cloning: laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory [ Cold spring harbor laboratory ], cold Spring Harbor (NY) [ Cold spring harbor (New York) ]; Silhavy et al (1984) Experiments with Gene Fusions [ Gene fusion experiments ], cold Spring Harbor Laboratory [ Cold spring harbor laboratory ], cold Spring Harbor (NY) [ Cold spring harbor (New York) ]; ausubel et al (1987) Current Protocols in Molecular Biology [ Current protocols in molecular biology ], greene Publishing assoc. And WILEY INTERSCIENCE [ Green publication Association and Willi science ]; Gelvin et al (eds.) (1990) Plant Molecular Biology Manual [ handbook of plant molecular biology ]; kluwer Academic Publisher [ g Lv Weier academy of sciences ], dordrecht, THE NETHERLANDS [ Dutch De Reich, tel.). However, further sequences, for example as linkers with specific cleavage sites for restriction enzymes or as signal peptides, may also be located between the two sequences. Insertion of the sequence may also result in expression of the fusion protein. Preferably, the expression construct consisting of a ligation of regulatory regions (e.g., a promoter and a nucleic acid sequence to be expressed) may be present in vector integrated form and inserted into the plant genome, e.g., by transformation.

Gene: the term "gene" refers to a region operably linked to suitable regulatory sequences capable of regulating the expression of a gene product (e.g., a polypeptide or functional RNA) in some manner. Genes include untranslated regulatory regions (e.g., promoters, enhancers, repressors, etc.) of DNA before (upstream) and after (downstream) the coding region (open reading frame, ORF), and where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term "structural gene" as used herein is intended to mean a DNA sequence transcribed into mRNA which is then translated into an amino acid sequence specific for a particular polypeptide.

"Gene editing" as used herein means the introduction of a specific mutation at a specific location in the genome of a cell. Gene editing may be introduced by performing precise editing using more advanced techniques, for example using CRISPR CAS systems and donor DNA, or CRISPR CAS systems (WO 15133554,WO 17070632) associated with mutagenic activity such as deaminase.

Genomic and genomic DNA: the term "genome" or "genomic DNA" refers to heritable genetic information of a host organism. The genomic DNA includes DNA of the nucleus (also referred to as chromosomal DNA), but also DNA of plastids (e.g., chloroplasts) and other organelles (e.g., mitochondria). Preferably, the term genomic or genomic DNA refers to chromosomal DNA of the nucleus.

Heterologous: the term "heterologous" with respect to a nucleic acid molecule or DNA refers to a nucleic acid molecule that is operably linked to or is manipulated to become operably linked to a second nucleic acid molecule that is not operably linked to the second nucleic acid molecule in nature (e.g., in the genome of a WT plant) or is operably linked to the second nucleic acid molecule in nature (e.g., in the genome of a WT plant) at a different location or position.

Preferably, the term "heterologous" with respect to a nucleic acid molecule or DNA (e.g., NEENA) refers to a nucleic acid molecule that is operably linked or otherwise manipulated to become operably linked to a second nucleic acid molecule (e.g., a promoter) to which the nucleic acid molecule is not operably linked in nature.

Heterologous expression constructs comprising a nucleic acid molecule and one or more regulator nucleic acid molecules linked thereto, such as a promoter or a transcription termination signal, are for example constructs produced by experimental manipulation, wherein a) the nucleic acid molecule, or b) the regulator nucleic acid molecule, or c) both, i.e. (a) and (b), are not located in their natural (native) genetic environment or have been modified by experimental manipulation, examples of which are substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment refers to a natural chromosomal locus in the organism of origin or to the presence in a genomic library. In the case of genomic libraries, the natural genetic environment of the nucleic acid molecule sequence is preferably preserved, at least partially. The environment is flanked on at least one side by nucleic acid sequences and has a sequence of at least 50bp, preferably at least 500bp, particularly preferably at least 1,000bp, very particularly preferably at least 5,000bp in length. When a naturally-occurring expression construct (e.g., a naturally-occurring combination of a promoter and a corresponding gene) is modified by a non-natural, synthetic "artificial" method (e.g., such as mutagenesis), the naturally-occurring expression construct becomes a transgenic expression construct. Such a process has been described (U.S. Pat. No. 5,565,350; WO 00/15815). For example, a nucleic acid molecule encoding a protein is considered heterologous with respect to the promoter, which nucleic acid molecule is operably linked to a promoter that is not the native promoter of the molecule. Preferably, the heterologous DNA is not endogenous or naturally associated with the cell into which it is introduced, but is obtained from another cell or is synthetic. Heterologous DNA also includes DNA sequences that contain some modification of the endogenous DNA sequence, multiple copies of the endogenous DNA sequence that are not naturally occurring, or are not naturally related to another DNA sequence to which they are physically linked. Typically, although not necessarily, the heterologous DNA encodes RNA or a protein that is not normally produced by the cell expressing the DNA.

Hybridization: the term "hybridization" as defined herein is a process in which substantially complementary nucleotide sequences anneal to each other. The hybridization process may occur entirely in solution, i.e., both complementary nucleic acids are in solution. Hybridization can also occur where one of the complementary nucleic acids is immobilized to a substrate (e.g., a magnetic bead, agarose bead, or any other resin). Furthermore, the hybridization process can take place in the case of one of the complementary nucleic acids immobilized on a solid support such as nitrocellulose or nylon membrane or immobilized by, for example, photolithography on, for example, a siliceous glass support (the latter referred to as a nucleic acid array or microarray or as a nucleic acid chip). To allow hybridization to occur, nucleic acid molecules are typically thermally or chemically denatured to melt the double strand into two single strands and/or to remove hairpins or other secondary structures from the single stranded nucleic acid.

The term "stringency" refers to the conditions under which hybridization occurs. The stringency of hybridization is affected by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. Typically, the low stringency conditions are selected to be about 30 ℃ below the thermal melting point (Tm) of the particular sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is below Tm 20 ℃, while high stringency conditions are when the temperature is below Tm 10 ℃. High stringency hybridization conditions are typically used to isolate hybridization sequences that have high sequence similarity to the target nucleic acid sequence. However, due to the degeneracy of the genetic code, nucleic acids may deviate in sequence and still encode substantially the same polypeptide. Thus, medium stringency hybridization conditions may sometimes be required to identify such nucleic acid molecules.

"Tm" is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a perfectly matched probe. The Tm depends on the solution conditions and the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. The maximum rate of hybridization is achieved at about 16℃up to 32℃below the Tm. The presence of monovalent cations in the hybridization solution reduces electrostatic repulsion between the two nucleic acid strands, thereby promoting hybrid formation; this effect is visible for sodium concentrations up to 0.4M (for higher concentrations this effect is negligible). For each percentage of formamide, formamide lowers the melting temperature of the DNA-DNA and DNA-RNA duplex by 0.6 ℃ to 0.7 ℃, and the addition of 50% formamide allows hybridization at 30 ℃ to 45 ℃, although the hybridization rate would decrease. Base pair mismatches reduce the hybridization rate and thermal stability of the duplex. On average and for large probes, the Tm is reduced by about 1 ℃/% base mismatches. Depending on the type of hybrid, tm can be calculated using the following equation:

DNA-DNA hybrids (Meinkoth and Wahl, anal. Biochem. [ analytical biochemistry ],138:267-284,1984):

Tm=81.5deg.C+16.6xlog [ Na+ ] a+0.41x% [ G/Cb ] -500x [ lc ] -1-0.61x% carboxamide

DNA-RNA or RNA-RNA hybrids:

Tm＝79.8+18.5(log10[Na+]a)+0.58(％G/Cb)+11.8(％G/Cb)2-820/Lc

oligo-DNA or oligo-RNAd hybrid:

for <20 nucleotides: tm=2 (ln)

For 20-35 nucleotides: tm=22+1.46 (ln)

A or for other monovalent cations, but only in the range of 0.01-0.4M.

B for% GC, accuracy is only in the range of 30% to 75%.

C L = length of duplex in base pair.

D Oligo, oligonucleotide; ln, effective length of primer = 2× (number of G/C) + (number of a/T).

Nonspecific binding can be controlled using any of a number of known techniques (such as, for example, blocking the membrane with a solution containing the protein, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase). For non-related probes, a series of hybridizations can be performed by changing one of the following: (i) Gradually decreasing the annealing temperature (e.g., from 68 ℃ to 42 ℃) or (ii) gradually decreasing the formamide concentration (e.g., from 50% to 0%). The skilled artisan is aware of various parameters that may be varied during hybridization, maintaining or altering stringent conditions.

In addition to hybridization conditions, the specificity of hybridization typically depends on the function of post-hybridization washes. To remove background from non-specific hybridization, the samples were washed with dilute saline solution. Key factors for such washing include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the washing temperature, the higher the stringency of the washing. Washing conditions are typically performed at or below hybridization stringency. Positive hybridization produces a signal that is at least twice that of the background signal. In general, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as described above. More stringent or less stringent conditions may also be selected. The skilled artisan is aware of various parameters that may be varied during washing, maintaining or changing stringency conditions.

For example, typical high stringency hybridization conditions for DNA hybrids longer than 50 nucleotides encompass hybridization in 1x SSC at 65 ℃ or hybridization in 1x SSC and 50% formamide at 42 ℃ followed by washing in 0.3x SSC at 65 ℃. Examples of moderately stringent hybridization conditions for DNA hybrids longer than 50 nucleotides encompass hybridization in 4x SSC at 50 ℃ or in 6x SSC and 50% formamide at 40 ℃ followed by washing in 2x SSC at 50 ℃. The length of the hybrid is the desired length for hybridization of the nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences and identifying the conserved regions described herein. 1 XSSC is 0.15M NaCl and 15mM sodium citrate; the hybridization and wash solutions may additionally comprise 5X Deng Hate reagents (Denhardt's reagent), 0.5% -1.0% SDS, 100. Mu.g/ml denatured fragmented salmon sperm DNA, 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridization in 0.1 XSSC containing 0.1SDS and optionally 5 XSSC at 65℃with 100. Mu.g/ml denatured fragmented salmon sperm DNA, 0.5% sodium pyrophosphate followed by washing in 0.3 XSSC at 65 ℃.

For the purpose of defining the level of stringency, reference can be made to Sambrook et al (2001) Molecular Cloning: a laboratory manual [ molecular cloning: laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press [ Cold spring harbor laboratory Press ], CSH, new York [ Cold spring harbor, N.Y. ], or reference Current Protocols in Molecular Biology [ Current molecular biology protocols ], john Wiley & Sons [ John Willi father, inc. ], N.Y [ New York ] (New year, 1989).

Indels are terms of random insertions or deletions of bases in the genome of an organism that are associated with NHEJ repair DSBs. It is classified as belonging to small genetic variations, measuring 1 to 10 000 base pairs in length. As used herein, it refers to random insertions or deletions of bases in or near the target site (e.g., less than 1000bp、900bp、800bp、700bp、600bp、500bp、400bp、300bp、250bp、200bp、150bp、100bp、50bp、40bp、30bp、25bp、20bp、15bp、10bp or 5bp upstream and/or downstream of the target site).

The terms "introducing (introducing)", "introducing (introduction)", etc. in reference to introducing a donor DNA molecule at a target site of a target DNA mean any introduction of a sequence of the donor DNA molecule into a target region, for example, by physically integrating the donor DNA molecule or a portion thereof into the target region or introducing the sequence of the donor DNA molecule or a portion thereof into the target region using the donor DNA as a template for a polymerase.

Introns: refers to the portion of DNA within a gene that does not encode a portion of the protein produced by the gene and that is spliced out of mRNA transcribed from the gene before the mRNA is exported from the nucleus (intervening sequence). An intron sequence refers to the nucleic acid sequence of an intron. Thus, introns are those regions of a DNA sequence that are transcribed together with the coding sequence (exon) but are removed during formation of the mature mRNA. Introns may be located within the actual coding region or in the 5 'or 3' untranslated leader sequence of the pre-mRNA (non-spliced mRNA). Introns in the primary transcript are excised and the coding sequences are simultaneously and precisely ligated to form the mature mRNA. The junction of the intron and exon forms a splice site. The sequence of the intron starts with GU and ends with AG. Furthermore, in plants, two examples of AU-AC introns have been described: the fourteenth intron of the RecA-like protein gene from arabidopsis thaliana (Arabidopsis thaliana) and the seventh intron of the G5 gene are AT-AC introns. The intron-containing pre-mRNA has three short sequences, which are necessary for the intron to be spliced accurately, among other sequences. These sequences are the 5 'splice site, the 3' splice site and the branch point. mRNA splicing is the removal of intervening sequences (introns) present in the primary mRNA transcript and the ligation of (joining or ligation) exon sequences. This is also known as cis-splicing, which links two exons on the same RNA, while removing intervening sequences (introns). The functional elements of an intron comprise sequences that are recognized and bound by the specific protein component of the spliced body (e.g., splice consensus sequences at the ends of the intron). Interaction of the functional element with the spliceosome results in removal of the intron sequence from the immature mRNA and reconnection of the exon sequence. Introns have three short sequences that are necessary, although not sufficient, for the intron to be spliced accurately. These sequences are the 5 'splice site, the 3' splice site and the branch point. The branch point sequence is important in plant splicing and splice site selection. The branch point sequence is typically located 10-60 nucleotides upstream of the 3' splice site.

Separating: the term "isolated" as used herein means a material that has been manually removed and that exists from its original natural environment and is therefore not a natural product. The isolated material or molecule (e.g., DNA molecule or enzyme) may be present in purified form or may be present in a non-natural environment (e.g., such as in a transgenic host cell). For example, a naturally occurring polynucleotide or polypeptide present in a living plant is not isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition and will be isolated in that such vector or composition is not part of its original environment. Preferably, the term "isolated" when used in reference to a nucleic acid molecule (as in "isolated nucleic acid sequence") refers to a nucleic acid sequence identified and isolated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated nucleic acid molecule is a nucleic acid molecule that exists in a form or environment that is different from the form or environment in which it is found in nature. In contrast, non-isolated nucleic acid molecules are nucleic acid molecules, such as DNA and RNA, that are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found adjacent to an adjacent gene on the host cell chromosome; RNA sequences (e.g., specific mRNA sequences encoding specific proteins) are found in cells as mixtures with a variety of other mrnas encoding a variety of proteins. However, an isolated nucleic acid sequence comprising, for example, SEQ ID NO. 1, for example, includes a nucleic acid sequence that normally comprises SEQ ID NO. 1 in a cell, wherein the nucleic acid sequence is at a different chromosomal or extra-chromosomal location than in a natural cell, or is otherwise flanked by nucleic acid sequences that are different from naturally found nucleic acid sequences. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is used to express a protein, the nucleic acid sequence will contain at least a portion of the sense strand or coding strand at a minimum (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both a sense strand and an antisense strand (i.e., the nucleic acid sequence may be double-stranded).

Non-coding: the term "non-coding" refers to a sequence of a nucleic acid molecule that does not encode part or all of the expressed protein. Non-coding sequences include, but are not limited to, introns, enhancers, promoter regions, 3 'untranslated regions, and 5' untranslated regions.

Nucleic Acid (NEENA) to enhance nucleic acid expression: the term "nucleic acid that enhances expression of a nucleic acid" refers to a sequence and/or nucleic acid molecule having a specific sequence that enhances the inherent properties of nucleic acid expression under the control of a promoter functionally linked to NEENA. Unlike the promoter sequence, NEENA itself cannot drive expression. In order to achieve the function of enhancing expression of nucleic acid molecules functionally linked to NEENA, NEENA itself must be functionally linked to a promoter. Unlike enhancer sequences known in the art, NEENA acts in cis rather than trans and must be located near the transcription initiation site of the nucleic acid to be expressed.

Nucleic acids and nucleotides: the terms "nucleic acid" and "nucleotide" refer to a naturally occurring or synthetic or artificial nucleic acid or nucleotide. The terms "nucleic acid" and "nucleotide" include deoxyribonucleotides or ribonucleotides or any nucleotide analogs and polymers, or hybrids thereof in either single-or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), and the complement as well as the sequence explicitly indicated. The term "nucleic acid" is used interchangeably herein with "gene," cDNA, "" mRNA, "" oligonucleotide, "and" polynucleotide. Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-pyrimidine modifications, 8-purine modifications, modifications on the exocyclic amine of cytosine, substitutions of 5-bromo-uracil, and the like; and sugar modifications at the 2 '-position, including but not limited to sugar modified ribonucleotides, wherein the 2' -OH is replaced by a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2 OR CN. Short hairpin RNAs (shrnas) may also comprise non-natural elements, such as non-natural bases (e.g., ionosin and xanthines), non-natural sugars (e.g., 2' -methoxyribose), or non-natural phosphodiester linkages (e.g., methylphosphonates, phosphorothioates, and peptides).

Nucleic acid sequence: the phrase "nucleic acid sequence" refers to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'terminus to the 3' terminus. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA, and DNA or RNA that plays a major structural role. "nucleic acid sequence" also refers to a continuous list of abbreviations, letters, characters or words representing nucleotides. In one embodiment, the nucleic acid may be a "probe," which is a relatively short nucleic acid, typically less than 100 nucleotides in length. Typically, the nucleic acid probe is about 50 nucleotides to about 10 nucleotides in length. A "target region" of a nucleic acid is a portion of the nucleic acid that is identified as the object. A "coding region" of a nucleic acid is that portion of the nucleic acid that, when placed under the control of appropriate regulatory sequences, is transcribed and translated in a sequence-specific manner to produce a particular polypeptide or protein. The coding region is considered to encode such a polypeptide or protein.

Nucleic acid molecules: the term "nucleic acid molecule" means a molecule comprising "nucleic acids" and "nucleotides". The term "nucleic acid molecule" preferably refers to small nucleic acid molecules, oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimics thereof, as well as functionally similar small nucleic acid molecules, polynucleotides or oligonucleotides having non-naturally occurring portions. Such modified or substituted small nucleic acid molecules, polynucleotides or oligonucleotides are generally preferred over the native form because they have desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases. A small nucleic acid molecule, polynucleotide, or oligonucleotide preferably comprises two or more core monomers covalently coupled to each other by a linkage (e.g., phosphodiester) or alternatively a linkage. For example, the term "nucleic acid molecule" refers to a DNA or RNA molecule, and is not limited to a particular "nucleic acid sequence. For example, the term "nucleic acid molecule" relates to molecules comprising the sequence of naturally occurring nucleic acid molecules or nucleotides and modifications thereof. The term includes molecules comprising naturally occurring sequences, e.g., sequences modified by the methods described herein. In one embodiment, the nucleic acid molecule is an oligonucleotide or polynucleotide.

An oligonucleotide: the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or a mimetic thereof, as well as functionally similar oligonucleotides having non-naturally occurring portions. Such modified or substituted oligonucleotides are generally preferred over the natural form because they have desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases. The oligonucleotide preferably comprises two or more core monomers covalently coupled to each other by a linkage (e.g., phosphodiester) or alternatively a linkage.

Protruding ends: an "overhang" is a relatively short single-stranded nucleotide sequence (also referred to as an "extension", "protruding end" or "sticky end") at the 5 '-or 3' -hydroxyl end of a double-stranded oligonucleotide molecule.

And (3) plants: is generally understood to mean any eukaryotic single-or multicellular organism capable of photosynthesis, or cells, tissues, organs, parts or propagation material thereof (such as seeds or fruits). For the purposes of the present invention, all genera and species of higher and lower plants of the kingdom Phytophthora are included. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. The term includes mature plants, seeds, shoots and seedlings, and derived parts thereof, propagation material (such as seeds or microspores), plant organs, tissues, protoplasts, calli and other cultures (e.g., cell cultures), and any other type of plant cell classified as producing a functional or structural unit. mature plants refer to plants that are at any desired developmental stage beyond the seedling. Seedlings refer to young immature plants at an early developmental stage. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred host organisms for the generation of transgenic plants. In addition, it is advantageous to express genes in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. Plants that may be mentioned by way of example and not by way of limitation are angiosperms; mosses, such as, for example, mosses (Hepaticae) (mosses) and mosses (Musci) (mosses); ferns, such as ferns, horsetail and pinus koraiensis; Gymnosperms, such as conifers, threes, ginkgo and Gnetum (Gnetidae); algae such as Chlorophyceae (Chlorophyceae), phaeophyceae (Phaeophyceae), rhodophyceae (Rhodophyceae), cyanophyceae (Myxophyceae), xanthophyceae (Xanthophyceae), diatomaceae (Baciliaropyceae) (diatoms) and Euglena (Euglenophyceae). Preferred are plants for food or feed purposes, such as leguminous (Leguminosae), such as peas, alfalfa and soybeans; Gramineae (Gramineae) such as rice, maize, wheat, barley, sorghum, millet, rye, triticale or oats; umbelliferae (Umbelliferae), especially Daucus, very especially the species Daucus carota/carrot, and Apium (Apium), very especially the species Apium graveolens (Apium Graveolens dulce) (celery) and many other plants; solanaceae (Solanaceae), especially Lycopersicon (Lycopersicon), very especially the species Lycopersicon (Lycopersicon esculentum) (tomato), and Solanum (Solanum), very especially the species Solanum tuberosum (Solanum tuberosum) (potato) and Solanum tuberosum (Solanum melongena) (eggplant) and many other plants (e.g. tobacco); And Capsicum (Capsicum), very particularly the species Capsicum annuum/pepper, and many other plants; leguminous (Leguminosae), especially Glycine (Glycine), very especially the species Glycine max/soybean, alfalfa, pea, alfalfa (lucerne), beans or peanuts, and many other plants; and Cruciferae (Cruciferae/Brassicacae), especially Brassica, very especially the species Brassica napus (rape), brassica napus (Brassica campestris) (beet (beet)), brassica oleracea Tastine cultivar (Brassica oleracea cv Tastie) (cabbage), brassica oleracea snowy cultivar (Brassica oleracea cv Snowball Y (broccoli) and Brassica oleracea cultivar (Brassica oleracea cv Emperor) (broccoli); And Arabidopsis (Arabidopsis), very particularly the species Arabidopsis (Arabidopsis thaliana) and many other plants; the family Compositae (Compositae), in particular the genus Lactuca (Lactuca), very particularly the species Lactuca sativa (lettuce) and many other plants; asteraceae (Asteraceae) such as sunflower, marigold, lettuce or calendula and many other plants; cucurbitaceae (Cucurbitaceae) such as melon, pumpkin (pumpkin/squash), or zucchini and flaxseed. Further preferred are cotton, sugarcane, hemp, flax, capsicum (chillies), and various tree, nut and vine species.

Polypeptide: the terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.

Pre-protein: proteins that are generally targeted to an organelle (e.g., a chloroplast) and still contain transit peptides thereof.

By "precise" with respect to introducing a donor DNA molecule in a target region is meant that the sequence of the donor DNA molecule is introduced into the target region without any indels, duplications or other mutations, as compared to the unaltered DNA sequence of the target region not comprised in the sequence of the donor DNA molecule.

Primary transcripts: the term "primary transcript" as used herein refers to an immature RNA transcript of a gene. "Primary transcripts" for example still contain introns, and/or do not yet contain multiple A-tails or cap structures, and/or lack other modifications (such as, for example, pruning or editing) necessary for proper functioning as transcripts.

Promoter: the term "promoter" or "promoter sequence" is equivalent and, as used herein, refers to a DNA sequence that is capable of controlling transcription of a nucleotide sequence of interest into RNA when linked to the nucleotide sequence of interest. Such promoters are found, for example, in the following public database ：http://www.grassius.org/grasspromdb.html ;http://mendel.cs.rhul.ac.uk/mendel.phptopic＝plantprom ;http://ppdb.gene.nagoya-u.ac.jp/cgi-bin/index.cgi. where the listed promoters are useful in the methods of the present invention and are included herein by reference. The promoter is located 5' (i.e., upstream) of the nucleotide sequence of interest under its control for transcription into mRNA, adjacent to the transcription initiation site, and provides a site for specific binding by RNA polymerase and other transcription factors to initiate transcription. The promoter comprises, for example, at least 10kb, for example 5kb or 2kb, adjacent to the transcription initiation site. It may also comprise at least 1500bp, preferably at least 1000bp, more preferably at least 500bp, even more preferably at least 400bp, at least 300bp, at least 200bp or at least 100bp adjacent to the transcription start site. In a further preferred embodiment, the promoter comprises at least 50bp, e.g. at least 25bp, adjacent to the transcription start site. Promoters do not contain exons and/or intronic regions or 5' untranslated regions. Promoters may, for example, be heterologous or homologous to the corresponding plant. A polynucleotide sequence is "heterologous" to an organism or second polynucleotide sequence if it originates from a foreign species, or if it originates from the same species but is modified relative to its original form. For example, a promoter operably linked to a heterologous coding sequence means that the coding sequence is from a species other than the species from which the promoter is derived, or if from the same species, the coding sequence (e.g., a genetically engineered coding sequence or an allele thereof from a different ecotype or variant) is not naturally associated with the promoter. Suitable promoters may be derived from genes of the host cell in which expression should occur, or from pathogens of such host cells (e.g., plants or plant pathogens such as plant viruses). Plant-specific promoters are promoters suitable for regulating expression in plants. It may be derived from plants, or from plant pathogens, or it may be a synthetic promoter designed by man. If the promoter is an inducible promoter, the transcription rate increases in response to the inducer. Likewise, a promoter may be regulated in a tissue-specific or tissue-preferred manner such that it is active only or primarily in transcribing relevant coding regions in one or more specific tissue types (e.g., leaf, root, or meristem). The term "tissue-specific" when applied to a promoter refers to a promoter capable of directing the selective expression of a nucleotide sequence of interest for a particular tissue type (e.g., petals), while the same nucleotide of interest is relatively absent from expression in a different type of tissue (e.g., root). The tissue specificity of a promoter may be assessed, for example, by operably linking a reporter gene to a promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into each tissue of the resulting transgenic plant, and detecting expression of the reporter gene in a different tissue of the transgenic plant (e.g., detecting mRNA, protein, or activity of the protein encoded by the reporter gene). The detection of a higher level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues indicates that the promoter is specific for the tissue in which the higher level of expression was detected. The term "cell type specific" when applied to a promoter refers to a promoter capable of directing the selective expression of a nucleotide sequence of interest in a particular type of cell, while the same nucleotide sequence of interest is relatively absent from expression in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means that the promoter is capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of the promoters can be assessed using methods well known in the art (e.g., GUS activity staining, GFP protein or immunohistochemical staining). The term "constitutive" when used in reference to a promoter or expression derived from a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid molecule in most plant tissues and cells in the substantial entirety of the plant or plant part's life in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). typically, constitutive promoters are capable of directing expression of a transgene in essentially any cell and any tissue.

Promoter specificity: the term "specific" when referring to a promoter means the expression pattern conferred by the corresponding promoter. The tissue and/or developmental status of a plant or part thereof is specifically described, wherein the promoter confers expression of the nucleic acid molecule under the control of the corresponding promoter. The specificity of a promoter may also include environmental conditions under which the promoter may be activated or down-regulated, such as induced or repressed by biological or environmental stresses (e.g., cold, drought, trauma, or infection).

And (3) purifying: as used herein, the term "purified" refers to a molecule (nucleic acid or amino acid sequence) that is removed, isolated, or separated from its natural environment. A "substantially purified" molecule is at least 60% free, preferably at least 75% free, and more preferably at least 90% free of other components with which it is naturally associated. The purified nucleic acid sequence may be an isolated nucleic acid sequence.

Recombination: the term "recombinant" with respect to nucleic acid molecules refers to nucleic acid molecules produced by recombinant DNA techniques. Recombinant nucleic acid molecules may also comprise molecules that are not naturally occurring themselves, but are modified, altered, mutated or otherwise manipulated by humans. Preferably, a "recombinant nucleic acid molecule" is a non-naturally occurring nucleic acid molecule that differs from the sequence of a naturally occurring nucleic acid molecule by at least one nucleic acid. "recombinant nucleic acid molecule" may also include "recombinant constructs" comprising nucleic acid molecule sequences, preferably operably linked, that do not occur naturally in that order. Preferred methods for producing the recombinant nucleic acid molecules may include cloning techniques, directed or non-directed mutagenesis, synthesis or recombinant techniques.

Significantly increasing or decreasing: for example, the increase or decrease in enzyme activity or gene expression is greater than the error range inherent in the measurement technique, preferably by about 2-fold or more, more preferably by about 5-fold or more, and most preferably by about 10-fold or more, of the control enzyme activity or expression in the control cell.

Small nucleic acid molecules: a "small nucleic acid molecule" is understood to be a molecule consisting of a nucleic acid or a derivative thereof, such as RNA or DNA. They may be double-stranded or single-stranded, and are between about 15 and about 30bp (e.g., between 15 and 30 bp), more preferably between about 19 and about 26bp (e.g., between 19 and 26 bp), even more preferably between about 20 and about 25bp (e.g., between 20 and 25 bp). In a particularly preferred embodiment, the oligonucleotide is between about 21 and about 24bp, for example between 21 and 24bp. In the most preferred embodiment, the small nucleic acid molecules are about 21bp and about 24bp, e.g., 21bp and 24bp.

Substantially complementary: in its broadest sense, the term "substantially complementary" when used herein with respect to a nucleotide sequence associated with a reference or target nucleotide sequence means a nucleotide sequence having a percent identity of at least 60%, more desirably at least 70%, more desirably at least 80% or 85%, preferably at least 90%, more preferably at least 93%, still more preferably at least 95% or 96%, yet still more preferably at least 97% or 98%, yet still more preferably at least 99% or most preferably 100% (in this context, the latter is equivalent to the term "identical") between the substantially complementary nucleotide sequence and the exact complementary sequence of the reference or target nucleotide sequence. Preferably, identity to the reference sequence is assessed over the entire length of at least 19 nucleotides, preferably at least 50 nucleotides, more preferably a nucleic acid sequence (if not otherwise specified below). Sequence comparisons were performed using the default GAP analysis of the SEQWEB application program of the university of Wisconsin GCG, GAP based on the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J mol. Biol. [ journal of molecular biology ]48:443-453; as defined above). A nucleotide sequence that is "substantially complementary" to a reference nucleotide sequence is hybridized to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions (as defined above).

"Target region" as used herein means a region that is close to, for example, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 100 bases, 125 bases, 150 bases, 200 bases, or 500 bases or more from a target site; or a region comprising a target site at which the sequence of the donor DNA molecule is introduced into the cell genome.

As used herein, "target site" means a location in the genome at which a double-strand break or one or a pair of single-strand breaks (nicks) are induced using recombinant techniques (e.g., zn-finger, TALEN, restriction enzyme, homing endonuclease, RNA-guided nuclease, RNA-guided nicking enzyme (e.g., CRISPR/Cas nuclease or nicking enzyme), etc.).

Transgenic: the term "transgene" as used herein refers to any nucleic acid sequence that is introduced into the genome of a cell by experimental manipulations. The transgene may be an "endogenous DNA sequence" or a "heterologous DNA sequence" (i.e., "exogenous DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence that naturally occurs in the cell into which it is introduced, so long as it does not contain some modifications (e.g., point mutations, the presence of a selectable marker gene, etc.) relative to the naturally occurring sequence.

Transgenic: the term transgene when referring to an organism means transformed, preferably stably transformed, with a recombinant DNA molecule preferably comprising a suitable promoter operably linked to a DNA sequence of interest.

And (3) a carrier: as used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto. One type of vector is a genomic integrative vector, or "integrative vector", which may integrate into the chromosomal DNA of the host cell. Another type of vector is episomal, i.e., a nucleic acid molecule capable of extrachromosomal replication. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors". In this specification, "plasmid" and "vector" are used interchangeably unless the context clearly indicates otherwise. Expression vectors designed to produce RNA as described herein in vitro or in vivo may contain sequences recognized by any RNA polymerase, including mitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III. According to the invention, these vectors can be used for transcription of the desired RNA molecules in cells. Plant transformation vectors are understood to be vectors suitable for plant transformation processes.

Wild type: the term "wild-type", "natural" or "natural origin" in reference to an organism, polypeptide or nucleic acid sequence means that the organism is naturally occurring or is available in at least one naturally occurring organism that has not been altered, mutated or otherwise manipulated by man.

According to one embodiment of the invention, the desired nucleic acid sequence is an RNA sequence. Thus, the method of the invention comprises a step allowing analysis of the RNA sequence. For example, RNA molecules are extracted and the frequency of the desired nucleic acid sequence in the RNA molecule is determined.

The methods of the invention may also include extracting RNA or RNA and DNA molecules, and generating DNA molecules from the extracted RNA molecules (e.g., using a reverse transcription method). The resulting DNA molecules or the resulting DNA molecules and the extracted DNA molecules are analyzed to determine the amount of the desired nucleic acid sequence. For example, RNA is extracted from regenerated cells or cultured calli or regenerated shoots or any other material to be analyzed according to the method of the invention.

Table:

Table 1: percentage of ddPCR mlo shedding (drop-off) of immature embryos from TMTA0233

Table 2: summary of mlo ddPCR results obtained for pooled leaf samples from TMTA0233

Table 3: summary of mlo ddPCR results obtained for individual shoots from TMTA0233

Table 4: summary of mlo NGS results obtained for a single analyzed shoot from TMTA 0233.

Annotation:

A. some of the individual alleles of the B and D mlo subgenomic cannot be analysed completely, for example +1 bp/indel. Such plants are double allelic mutants, or alternatively, one of the alleles carries a large indel sequence that interferes with NGS analysis. Shaded cells indicate plants with unique mutational characteristics.

Table 5: summary of mlo NGS results obtained for a single bud of one single embryo explant origin of TMTA0233

Annotation:

Table 6: percentage of ddPCR mlo shedding (drop-off) of immature embryos from TMTA0527

Table 7: summary of mlo ddPCR results obtained for pooled leaf samples from TMTA0527

Table 8: summary of mlo ddPCR results obtained for individual shoots from TMTA0527

Table 9: summary of mlo NGS results obtained for a single analyzed shoot from TMTA0527

Annotation:

A. some of the individual alleles of the B and D mlo subgenomic groups cannot be analysed completely, for example-6 bp/indel. Such plants are double allelic mutants, or alternatively, one of the alleles carries a large indel sequence that interferes with NGS analysis

Shaded cells indicate plants with unique mutational characteristics.

Table 10: summary of mlo NGS results obtained for a single bud of one single embryo explant origin of TMTA0527

Annotation:

A. some of the individual alleles of the B and D mlo subgenomic groups cannot be analysed completely, for example-9 bp/indel. Such plants are double allelic mutants, or alternatively, one of the alleles carries a large indel sequence that interferes with NGS analysis. Shaded cells indicate plants with unique mutational characteristics.

Table 11: summary of mlo NGS results obtained for offspring derived from plants TMTA-032-B01-06.

Table 12: summary of mlo NGS results obtained for offspring derived from plants TMTA-035-B04-01.

Table 13: summary of mlo NGS results obtained for offspring derived from plants TMTA-0233-044-B03-13.

Table 14: percentage of ddPCR FLC shedding from TMTA0609 immature embryos.

Table 15: summary of FLC ddPCR results obtained for pooled leaf samples from TMTA 0609.

Table 16: summary of FLC ddPCR results obtained for single shoots from TMTA 0609.

Drawings

Fig. 1: multiple sequence alignment depicting the position of Cas9 gRNA in exon 4 of three wheat mlo homologs. PAM sites are shown in boxes and gRNA recognition sites are underlined. Single nucleotide polymorphisms are shown in bold.

Fig. 2: schematic representation of ddPCR design, indicating the position, orientation and sequence of the different probes and primers. The predicted Cas9 cleavage site and the position of R158Q editing are also indicated. The editing probe binds only to the HDR modified allele, while the shedding probe loses its binding site when the NHEJ makes an insertion or deletion.

Fig. 3: verification of ddPCR assay by NGS analysis

Fig. 4: sensitivity was determined by ddPCR determined by serial dilution of HDR and NHEJ synthetic templates in constant background (200 ng) WT genomic DNA. The limit of detection of NHEJ was about 0.1% and the limit of detection of HDR was about 0.04% as determined by comparison with WT genomic DNA controls. Data are mean ± standard deviation of two pooled wells per dilution and three pooled wells of WT control.

Fig. 5: map of RNP bombarded immature wheat embryo region 1 (central target region), region 2 (inner loop target region) and region 3 (outer loop target region).

Fig. 6: leaf sampling procedure from the pool of PlantCon ^TM containers: shoots regenerated from callus derived from immature embryos (a), open containers showing shoots with long leaves (B), containers with shoots pre-trimmed prior to sampling (C), containers with shoots from which uniform leaf samples have been sampled (D), culture dishes with sampled pooled leaf explants (E), and containers ready for shoot regrowth and 6ml sample tubes (F).

Fig. 7: overview of the complete 3-step selection system for identifying RNP-induced deletion of insertions in wheat plants.

Fig. 8: detailed protocols for selection/analysis procedures for identifying Cas9 RNP-induced insertion deletions in wheat plants indicate the timelines and procedures from embryo RNP bombardment to transfer of structured embryogenic callus into PlantCon ^TM containers. The time points of sample 1 are indicated in bold.

Fig. 9: detailed protocols for selection/analysis procedures for identifying Cas9 RNP-induced deletion of insertions in wheat plants indicate the timelines and procedures from transfer of structured embryogenic callus into PlantCon ^TM containers to transfer of mutant plants into the greenhouse. Time points of sample 2 and sample 3 are indicated in bold.

Fig. 10: two-dimensional plots generated by Quantasoft software for mlo ddPCR analysis performed on immature embryo samples, TMTA0233-B01$001ddPCR shed 5.6% (A), TMTA0233-B02$001ddPCR shed 4.0% (B), and TMTA0233-B05$001ddPCR shed 5.0% (C). NHEJ (fam+, HEX-) =upper left quadrant, WT allele (fam+, hex+) =upper right quadrant, and negative microdroplet (FAM-, HEX-) =lower left quadrant.

Fig. 11: bar graphs showing the percentage categories of ddPCR mlo shedding obtained from the analysed bud pools from 3 TMTA shots (category <10% shedding, 10% -25% shedding, 25% -50% shedding and >50% shedding).

Fig. 12: two-dimensional plots of representative pools generated by Quantasoft software, which show the following ddPCR mlo shedding percentages: TMTA0233-045-B03$001, <10% (A); TMTA0233-058-B02$001, 10% -25% (B); TMTA0233-040-B02$001, 25% -50% (C); and TMTA-035-B03$001, >50% (D). NHEJ (fam+, HEX-) =upper left quadrant, WT allele (fam+, hex+) =upper right quadrant, and negative microdroplet (FAM-, HEX-) =lower left quadrant.

Fig. 13: two-dimensional plots of representative individual shoots generated by Quantasoft software, showing percentages of ddPCR mlo shedding of 0% (A), 50% (B) and 100% (C). NHEJ (fam+, HEX-) =upper left quadrant, WT allele (fam+, hex+) =upper right quadrant, and negative microdroplet (FAM-, HEX-) =lower left quadrant.

Fig. 14: PCR fragment size analysis of 86 individual plants (primer set Seq ID No:1/Seq ID No: 2). 6 plants from pool TMTA-035-B04, 25 plants from pool TMTA0233-037-B01, 14 plants from pool TMTA0233-037-B02, 16 plants from pool TMTA0233-044-B03 and 25 plants from pool TMTA0233-046-B02 were analyzed. Plants marked with arrows showed 0% abscission during ddPCR analysis.

Fig. 15: multiple sequence alignment depicting the position of Cas12a gRNA in exon 4 of three wheat mlo homologs. PAM sites are shown in boxes and gRNA recognition sites are underlined. Single nucleotide polymorphisms are shown in bold.

Fig. 16: schematic representation of ddPCR design, indicating the position, orientation and sequence of the different probes and primers. The predicted Cas12a cleavage site and the position of R158Q editing are also indicated. The editing probe binds only to the HDR modified allele, while the shedding probe loses its binding site when the NHEJ makes an insertion or deletion.

Fig. 17: detailed protocols for selection/analysis procedures for identifying Cas12a RNP-induced insertion deletions in wheat plants indicate the timelines and procedures from embryo RNP bombardment to transfer of structured embryogenic callus into PlantCon ^TM containers. The time points of sample 1 are indicated in bold.

Fig. 18: detailed protocols for selection/analysis procedures for identifying Cas12a RNP-induced deletion of insertions in wheat plants indicate the timelines and procedures from transfer of structured embryogenic callus into PlantCon ^TM containers to transfer of mutant plants into the greenhouse. Time points of sample 2 and sample 3 are indicated in bold.

Fig. 19: two-dimensional plots generated by Quantasoft software for mlo ddPCR analysis performed on immature embryo samples, TMTA0527-B01$001ddPCR shed 1.9% (A), TMTA0527-B02$001ddPCR shed 2.7% (B), and TMTA0527-B05$001ddPCR shed 1.6% (C). NHEJ (fam+, HEX-) =upper left quadrant, WT allele (fam+, hex+) =upper right quadrant, and negative microdroplet (FAM-, HEX-) =lower left quadrant.

Fig. 20: two-dimensional plots of representative pools generated by Quantasoft software, which show the following ddPCR mlo shedding percentages: TMTA0527-031-B01$001, <10% (A); TMTA0527-033-B02$001, 10% -25% (B); TMTA0527-039-B04$001, 25% -50% (C); and TMTA-0527-044-B06$001, >50% (D). NHEJ (fam+, HEX-) =upper left quadrant, WT allele (fam+, hex+) =upper right quadrant, and negative microdroplet (FAM-, HEX-) =lower left quadrant.

Fig. 21: two-dimensional plots of representative individual shoots generated by Quantasoft software, showing percentages of ddPCR mlo shedding of 0% (A), 50% (B) and 100% (C). NHEJ (fam+, HEX-) =upper left quadrant, WT allele (fam+, hex+) =upper right quadrant, and negative microdroplet (FAM-, HEX-) =lower left quadrant.

Fig. 22: PCR fragment size analysis of individual plants recovered from different pools of TMTA0527 (primer combination Seq ID NO:1/Seq ID NO: 2).

Fig. 23: PCR fragment size analysis of 62 individual plants regenerated from single embryo explants (TMTA-0527-045) (primer combination Seq ID NO:1/Seq ID NO: 2). 18 plants from pool TMTA0527-045-B02, 13 plants from pool TMTA0527-045-B03, 16 plants from pools TMTA0527-045-B04 and TMTA0527-045-B05 were analyzed. Plants selected for NGS are indicated by arrows (mlo's subgenomic a alleles, previously characterized as 0%, 50% or 100% ddpcr abscission).

Fig. 24: sequence alignment depicting the position of Cas12a gRNA in 3 subgenomic copies of TaAGL (FLC). PAM sites are shown in boxes and gRNA recognition sites are underlined. Single nucleotide polymorphisms are shown in bold.

Fig. 25: schematic of FLC ddPCR design, indicating the position, orientation and sequence of the different probes and primers used to detect indels generated at Cas12a (cr-FLC-G1) cleavage site.

Fig. 26: distribution of RNP-targeted FLC alleles in recovered individual plants from 3 independent shots of experiment TMTA 0609.

Fig. 27: sequence(s)

Examples

Chemicals and methods of use

Cloning procedures for the purposes of the present invention, including restriction digestion, agarose gel electrophoresis, nucleic acid purification, nucleic acid ligation, transformation, selection and cultivation of bacterial cells are performed as described (Sambrook et al, 1989), unless otherwise indicated. Sequence analysis of recombinant DNA was performed using the Sanger technique (Sanger et al, 1977) with a laser fluorescence DNA sequencer (applied biosystems (Applied Biosystems), foster City, calif., U.S.A.). Unless otherwise described, chemicals and reagents were obtained from sigma-aldrich (SIGMA ALDRICH) (sigma-aldrich, st.s.i.), prolog (Promega) (madison, wisconsin, u.s.a.), duchefa (hallem, netherlands), or Invitrogen (Invitrogen, ca, u.s.a.). Restriction endonucleases were from new england biological laboratories (NEW ENGLAND Biolabs) (ibos wa, ma) or rogowski diagnostic, inc (Roche Diagnostics GmbH) (Peng Cibei grid, germany). Oligonucleotides were synthesized by European Union genome company (Eurofins Eurofins Genomics) (Epsburg, germany) or comprehensive DNA Technologies (INTEGRATED DNA Technologies) (Aiload Hua Zhouke Lerval, U.S.A.).

EXAMPLE 1-mlo Cas9 RNP targeting

Cas 9-targeted gRNA design for mlo

Based on previous work (Gil-Humanes et al 2017), we designed a synthetic crRNA targeting wheat mildew resistance locus O (mlo) consisting of a 16bp direct repeat and a 20bp pre-spacer. The mlo gene encodes a seven transmembrane domain protein which is involved in resistance to the fungal pathogen erysiphe graminis (Blumeria graminis). The recognition site of crRNA is located within exon 4 of the antisense strand of mlo [5'-GAACTGGTATTCCAAGGAGG (CGG) -3' ], with the PAM site between brackets. The designed guide was specific for the 5A and 4D alleles of mlo and showed a mismatch with the 4B allele at position 6 from the PAM sequence (fig. 1).

Design of a microdroplet digital PCR (ddPCR) assay to simultaneously identify Cas9 RNP-induced mlo indel mutations and Precisely edited

Current methods of detecting genome editing events include gel-based systems, artificial reporter assays, high resolution melting curve analysis, and next generation sequencing. Microdroplet digital PCR is a quick alternative to these methods, enabling rapid and systematic quantification of genome editing results at endogenous loci. In a microdroplet digital PCR system, each PCR sample is divided into a number of microdroplets. PCR amplification occurs simultaneously in each droplet. At the end of the run, each droplet was individually assessed for the presence (positive) or absence (negative) of a fluorescent signal. Using poisson statistical analysis, the ratio of positive droplets to negative droplets gives an absolute quantification of the initial copy number of the target sequence.

While current applications focus on detecting insertions and deletions, ddPCR assays were designed that were able to measure NHEJ and HDR at endogenous loci simultaneously. For this, we designed three types of probes, all within one amplicon (FIG. 2). The first (reference probe) was labeled with FAM and was located at a position remote from the mutagenesis site. This probe counts all genomic copies of the target. The second (so-called shedding probe) is labeled with HEX and is located where Cas9 nuclease cleaves the mlo target. If Cas9 induces NHEJ, the shedding probe loses its binding site, resulting in loss of HEX and leaving only the FAM signal of the reference probe. The third probe (also FAM-labeled) binds to the desired DNA edit, causing additional gain in FAM signal when the exact edit is introduced. Using this assay, indel mutations, WT alleles and precise editing can be detected in distinct, clearly separated droplets with high sensitivity and low background signal.

DdPCR assays were designed for mlo 5A alleles using Primer3Plus software, using modified settings compatible with the following premix: namely, 50mM monovalent cation, 3.0mM divalent cation and 0mM dNTP; and SantaLucia thermodynamic and salt correction parameters. The predicted nuclease cleavage site (3 bp from PAM) is located in the middle of the amplicon with 70-100bp flanking sequences on either side up to the primer binding site. To avoid losing the binding site, the primer and reference probe are designed to be remote from the cleavage site. In addition, dark, 3' -phosphorylated non-extendable oligonucleotides were designed to prevent binding of the editing probe to the WT sequence.

PCR primers were designed according to the following guidelines: the primer has a length of 17-24 bases, a melting temperature of 55-60 ℃ (wherein the ideal temperature is 58 ℃, the melting temperature of the two primers is not more than 2 ℃), the GC content of the primers is 35-65%, and the amplicon size is 100-250 bases.

The considerations for probe design are as follows: probes can bind to either strand of the target, the GC content of the probe is 35% -65%, the 5 'end is free of G to prevent quenching of the 5' fluorophore, the melting temperature of the shedding probe ranges from 61℃to 64 ℃ (where the ideal temperature is 62 ℃), the length of the shedding probe is less than 20 bases, the melting temperatures of the reference probe and the editing probe ranges from 63℃to 67 ℃ (where the ideal temperature is 65 ℃), and the lengths of the reference probe and the editing probe are 20-24 bases. Preferably, the probe should have a Tm that is 4℃to 8℃higher than the primer. Primers and probe designs were also screened for complementarity and secondary structure, where the maximum Δg value for any of the self-dimer, hairpin, and heterodimer was set to-9.0 kcal/mole. All primers and probes were designed for the 5A allele of the wheat mlo gene.

The optimal annealing temperature was empirically determined using temperature gradient PCR.

The synthetic dsDNA fragment (gBlock, integrated DNA technologies) was used as a positive control for assay validation, the HDR positive control contained an R158Q substitution at the desired editing site, while the NHEJ specific control had a 1bp insert at the predicted nuclease cleavage site. The lyophilized gBlock was resuspended in 300 μl TE and stored at-20deg.C. Three additional dilutions in TE resulted in approximately 600 copies/. Mu.l (quantitatively confirmed by ddPCR) of master stock. The high copy gBlock stock was kept in a post PCR environment to avoid contamination.

The 20 XddPCR mixture consisted of 18. Mu.M forward primer (Seq ID NO: 1) and 18. Mu.M reverse primer (Seq ID NO: 2), 5. Mu.M reference probe (Seq ID NO: 3), 5. Mu.M editing probe (Seq ID NO: 4), 5. Mu.M shedding probe (Seq ID NO: 5) and 10. Mu.M dark probe (Seq ID NO: 6). The following reagents were mixed in 96-well plates to perform a 25 μl reaction: mu.l of probe ddPCR Supermix (without dUTP), 1.1. Mu.l of 10 Xassay mix (Berle laboratories (BioRad Laboratories)), 10U HindIII-HF, 100-250ng of genomic DNA in water, and water (up to 22. Mu.l).

Droplets were generated using a QX100 droplet generator according to manufacturer's instructions (bure laboratories) and transferred into 96-well plates for standard PCR on a C1000 thermal cycler with deep hole blocks (bure laboratories).

The thermal cycle consists of: an activation period of 10min at 95 ℃ followed by a two-step thermal profile of 40 cycles (i.e., denaturation at 95 ℃ for 30s and combined annealing-extension at 60 ℃ for 3 min) and 98 ℃ for 1 cycle for 10min.

Following PCR, droplets were analyzed in "absolute quantification" mode using a QX100 droplet reader (berle laboratories). To achieve proper gating for precise editing and indel events, experiments were performed using both negative and positive controls (unmodified genomic DNA and gBlock containing the R158Q mutation, respectively). In the two-dimensional plot, droplets without template were gated as a negative population. Droplets containing only NHEJ (fam+, HEX-) only, only HDR allele (fam++, HEX-) or only WT allele (fam+, hex+) were manually gated as separate populations. Allele frequencies were quantified using QuantaSoft v.1.2.10.0 (bure laboratories).

The designed ddPCR assay was verified by Next Generation Sequencing (NGS) of the target region using a pair of primers specific for the A subgenomic allele of the wheat mlo gene (Seq ID NO:7/Seq ID NO: 8). The amplicon was purified and deep sequenced (2 x 250bp paired ends) using Illumina MiSeq system (Jin Weizhi Germany, inc. (GENEWIZ, germany, gmbH)). A very good correlation (r2=0.96) was observed between the indel allele frequencies detected by ddPCR and NGS in the different samples, demonstrating the sensitivity and reliability of the ddPCR assay (fig. 3).

To calculate the limit of detection of the ddPCR assay, we spiked different amounts of HDR and NHEJ specific gBlock (Seq ID NO:9/Seq ID NO: 10) in wild type genomic wheat DNA and found that the assay was reproducible and linear over a wide range of input DNA (FIG. 4). For NHEJ the limit of detection is about 0.1% and for HDR alleles the limit of detection is well below 0.04%. This suggests that at least one indel or exact editing event from 1,000 copies of the genome can be captured by this assay.

Wheat donor plant growth for immature embryo isolation

The donor plants of variety Fielder are grown under controlled environmental conditions to ensure optimal immature embryo quality. Typical growth conditions are day/night temperatures of 20 ℃ +/-1 ℃ and 18 ℃ +/-1 ℃, relative humidity of 65%, 16h photoperiod (400 μmol/m ²/s table level) illuminance provided by a mixture of 600W high pressure sodium lamp and 400W metal halide lamp.

Preparation of wheat immature embryos for bombardment

Immature seeds were harvested from donor plants containing embryos of about 2mm in length, peeled and sterilized in 70% v/v ethanol for 1min, then in 10% v/v sodium hypochlorite (ACROS disinfectant containing 10% -15% active chlorine) for 10min. Finally, the immature seeds are washed several times with demineralised water.

Embryos were aseptically excised from immature seeds using a binocular microscope (model MZ6, lycra company (Leica)), and the hypocotyls through the green seed coats were carefully excised during preparation. Subsequent steps of embryo preparation were performed using the modified procedure substantially as described in Ishida et al (2015). Immature embryo explants were transferred to a 3.5cm petri dish (Falcon) 351008 containing 4.5ml of non-selective callus induction medium WLS (referred to herein as callus induction medium 240). The embryos are arranged in a 1.5cm center circle (25-50 embryos/petri dish, scutellum side up).

Preparation of Cas9 RNP complexes

Purified Cas9 nuclease, universal 67mer tracerRNA, and mlo specific crRNA were ordered from IDT (integrated DNA technologies) for RNP assembly.

-CrRNA: custom and user-defined crRNA that binds to 20 bases on the DNA strand opposite NGG (PAM sequence)

-TracrRNA: universal 67mer transactivation crRNA (tracrRNA), which contains proprietary chemical modifications that confer increased nuclease resistance. It hybridizes to crRNA to activate Cas9 enzyme

Cas9 nuclease @S.p.cas9 nuclease 3 NLS): recombinant streptococcus pyogenes(s) Cas9 nuclease purified from an escherichia coli (e.coli) strain expressing codon optimized Cas 9. Contains 1N-terminal Nuclear Localization Sequence (NLS), 2C-terminal NLS and C-terminal 6-His tag.

For RNP complex assembly, mlo-specific crRNA and tracrRNA were mixed in equimolar amounts to give a final duplex concentration of about 4 μm. The RNA complex was heated to 95 ℃ in a heating block for 5min, cooled to room temperature, and mixed with Cas9 nuclease at equimolar concentration in Cas9 reaction buffer (10 x stock = 200mM HEPES,100mM MgCl26H2O,5mM DTT,1500mM KCl, prepared in rnase-free water, ph 7.5). The mixture was incubated at room temperature for 10min and then transferred to ice.

Cas9 RNP complex delivery to gene gun in wheat

Use substantially as described in Liang et al (2018)PDS-1000/He particle delivery System (Bio-Rad) delivers RNPs into immature embryos. The RNP complex was mixed with 0.6 μm gold particles (BioRad) and 15 μl aliquots were coated on the central area of each large carrier (macrocarrier) and air dried in a laminar flow bench for 30min before delivery. For each shot, 200 μg of gold particles and 2 μg of Cas9 protein complexed with crRNA were delivered.

Cultivation of bombarded wheat immature embryos and plant regeneration

Following bombardment, immature embryos are incubated on the same plate in the dark (25 ℃ +/-1 ℃ C., 55% relative humidity in MLR-352H-PE Panasonic incubator) for 24H and then transferred to fresh non-selective callus induction medium 240 (9 cm dishes containing 35-40ml medium, up to 15 embryos/dishes).

Eight days after bombardment, immature embryos are split longitudinally under a binocular microscope into 2 pieces and transferred to fresh non-selective callus induction medium 240 (9 cm dishes, 12 embryos/dish containing 35-40ml medium) and cultured under the same conditions in the dark.

After 2 weeks, the bisected immature embryos were bisected again under a binocular microscope and transferred to fresh non-selective callus induction medium 240 (9 cm dishes containing 35-40ml medium, 4-6 embryos/dish) and cultured in the dark under the same conditions. After a further 2 weeks, the calli from each embryo were transferred intact to fresh non-selective callus induction medium 240 (9 cm dishes containing 35-40ml medium, 2 embryos/dish) and cultured under the same conditions in the dark.

Two weeks later, small pieces of structured embryogenic callus were transferred to non-selective regeneration medium LSZ (referred to herein as regeneration medium 420) (Ishida et al 2015). Regeneration medium 420 was prepared in PlantConTMTM vessels (MP biomedical, cat. No. 26-722-06), 100ml medium/vessel. Embryogenic callus produced from one embryo was transferred to one PlantConTM container (up to 16 pieces per container). In case more than 16 embryogenic calli are recovered from one embryo, an additional regeneration vessel is utilized. The PlantConTM vessels were incubated under light (23 ℃ +/-1 ℃ for 16h photoperiod) for approximately 6 weeks.

Shoots from PlantConTM containers were transferred individually to De Wit tubes (Duchefa Biochemie company) containing 10ml of non-selective rooting medium WRM (essentially a modification of medium R) (Sparks and Jones, 2009), solidified with 0.15% w/v Gelrite (Duchefa Biochemie company) and cultured under light (23 ℃ +/-1 ℃ C., 16h photoperiod). The rooted plants were transferred to a greenhouse.

Tissue sampling and DNA isolation

Two days after sampling 1-bombardment, 5 immature embryos were randomly selected from region 2 (fig. 5) of each bombarded plate. Expanding the embryo as a sample, and collecting in a 2ml tubeSafe-Lock) and stored at-80 ℃. The samples were ground in a Retsch hybrid mill MM300/400 for 60s. Genomic DNA extraction was performed using QIAGEN DNEASY plant mini kit (catalog No. 69106) according to the manufacturer's instructions. The final DNA concentration was measured and the plates were stored at 4 ℃ until used for downstream analysis.

Sampling 2-leaves were harvested from regeneration shoots after culturing the structured embryogenic callus on non-selective regeneration medium 420 in PlantCon ^TM vessels for 4 weeks. Care was taken to ensure that similarly sized leaves were obtained from all regenerated shoots, which was best achieved by first cutting all longer tips with sterile scissors, and then cutting leaves of approximately 5mm length from the remaining tissue (fig. 6). The leaves from each PlantCon ^TM containers were combined and transferred to a 6ml screw cap tube (michigan MP company (micron MP) 32301) and stored at-80 ℃. Samples were freeze-dried overnight and genomic DNA was extracted using a LGC GENOMICS sbeadexTM Maxi plant kit based procedure with KingFisher automation. The final DNA concentration was measured and the plates were stored at 4 ℃ until used for downstream analysis.

Sampling 3-leaves were harvested from shoots after 1-2 weeks of culture of individual shoots in De Wit tube containing 10ml WRM medium. Samples were collected in 1.4ml push-cap tubes in 96 sample carriers (Miehold MP company 226 RP) and stored at-80 ℃. Genomic DNA was extracted using a program based on LGC GENOMICS sbeadexTM Maxi plant kit and KingFisher automation. The final DNA concentration was measured and the plates were stored at 4 ℃ until used for downstream analysis.

Selection/analysis procedure to identify Cas9 RNP indels in mlo

FIG. 7 shows an overview of a 3-step selection system for identifying RNP-induced insert deletions in wheat plants mlo. This process involves sequential enrichment of tissues carrying RNP-induced mlo insertion deletions by repeated sampling and molecular screening to specifically detect targeted mutations. In the example shown, this is done in 3 steps in immature embryo explants, in a population of regenerated shoots, finally on a single plant level. A detailed overview of the selection/analysis process of Cas9 RNPs is shown in fig. 8 and 9.

Two days after gene gun RNP delivery, 5 immature embryos from each shot were selected from the inner loop region (region 2) and sampled for DNA isolation and ddPCR analysis to determine the percentage of mlo NHEJ shedding. The 20 XddPCR mixture consisted of 18. Mu.M forward primer (Seq ID NO: 1) and 18. Mu.M reverse primer (Seq ID NO: 2), 5. Mu.M reference probe (Seq ID NO: 3) and 5. Mu.M shedding probe (Seq ID NO: 5). The following reagents were mixed in 96-well plates to perform a 25 μl reaction: 11. Mu.l of probe ddPCR Supermix (without dUTP), 1.1. Mu.l of 10 Xassay mix (Berle laboratories Inc.), 100-250ng of genomic DNA in water, and water (up to 22. Mu.l). The results are summarized in table 1, with ddPCR shedding percentages ranging from 2.0% to 5.6%.

FIG. 10 shows a two-dimensional plot of immature embryo samples TMTA-B01 $001, TMTA0233-B02$001, and TMTA0233-B05$001 analyzed by ddPCR generated by Quantasoft software. The cluster seen in the upper left quadrant of each panel resulted from signals from amplicons representing NHEJ indels (RNP-targeted mlo alleles). However, the clusters seen in the upper right quadrant of each plot resulted from signals from amplicons representing WTs (non-RNP targeted mlo alleles). Samples TMTA-B01$001, TMTA0233-B02$001, and TMTA0233-B05$001 showed the highest percent shedding relative to all bombarded plates, thus further culturing the remaining embryos from these shots alone. ddPCR results indicate that these immature embryos are the most likely to carry cells with NHEJ indels in the mlo allele.

Immature embryos are cultured on non-selective callus induction medium 240 for 3 cycles to obtain structured embryogenic callus from which plants can be regenerated in PlantConTM vessels on non-selective regeneration medium 420. Leaves from multiple shoots in each PlantConTM containers were pooled and transferred to a 6ml screw cap tube (michaustorium MP company 32301) for ddPCR analysis to determine the percentage of mlo shedding. From the 3-culture shots of TMTA0233,023, a total of 173 pools from 62 individual embryos were analyzed (63 pools from shot 1, 78 pools from shot 2, and 32 pools from shot 5). For ease of presentation, the mlo shedding percentages for each pool are categorized into different categories (< 10%, 10% -25%, 25% -50% and > 50%). The results are shown in table 2 and fig. 11. Although varying greatly, each shot contained a pool that exhibited a high percentage of shedding (e.g., 1:63.5% pool exhibited a percentage of shedding of >25%, 2:32% pool exhibited a percentage of shedding of >25%, and 5:46.8% pool exhibited a percentage of shedding of > 25%).

FIG. 12 shows a two-dimensional plot of representative pools generated by Quantasoft software showing the percentage of ddPCR shedding of <10% (TMTA-0233-045-B03$001), 10% -25% (TMTA-0233-058-B02$001), 25% -50% (TMTA-0233-040-B02$001), and >50% (TMTA-0233-035-B03$001). The cluster seen in the upper left quadrant of each panel resulted from signals from amplicons representing NHEJ indels (RNP-targeted mlo alleles). However, the clusters seen in the upper right quadrant of each plot resulted from signals from amplicons representing WTs (non-targeted mlo alleles).

Shoots from pools exhibiting a percentage of shedding of >50% were mainly selected for further culture (13 PlantConTM containers from shoot 1). ddPCR results indicate that these pools are the most likely pools to carry shoots with NHEJ insertion deletions in the mlo allele. In addition, shoots from several other pools of shoot 1 (showing lower percent shedding) were also selected for comparison (5 PlantConTM containers with 25% -50% percent shedding, 6 containers with 10% -25% percent shedding and 2 containers with <10% percent shedding).

Shoots from selected containers were transferred to individual De Wit tubes containing 10ml of non-selective rooting medium WRM for further development. Approximately 2 weeks after transfer, leaf samples from individual plants were taken for ddPCR analysis to determine the percentage of mlo shedding. Samples were taken from 433 individual shoots from 17 immature embryo explants in total. Shoots were assigned to specific species based on the percentage of shedding obtained with ddPCR assays specifically designed for the a subgenomic group (i.e., near 0% value = WT, near 50% value = single allele mutation, and near 100% value = double allele mutation). The results of 426 shoots that can be unambiguously assigned to a particular species are shown (Table 3). For the other seven plants, a median percentage shedding was observed and could not be accurately assigned to any species. Of 426 plants categorized into different categories, 224 plants (53%) showed a percentage of abscission consistent with mlo RNP targeting: 95 plants had a percentage of abscission (=monoallelic) of 50%, and 129 plants had a percentage of abscission (=biallelic) of 100%. FIG. 13 shows a two-dimensional plot of representative plants generated by Quantasoft software for each ddPCR shed percentage species (0% shed: TMTA0233-032-B01-11, TMTA0233-044-B03-01, TMTA0233-046-B02-24, TMTA0233-047-B03-04;50% shed: TMTA0233-032-B02-03, TMTA0233-037-B01-04, TMTA0233-044-B03-15, TMTA0233-050-B04-08; and 100% shed: TMTA0233-032-B01-06, TMTA0233-035-B03-13, TMTA0233-037-B02-14, TMTA-047-B03-05). The cluster seen in the upper left quadrant of each panel resulted from signals from amplicons representing NHEJ indels (RNP-targeted mlo alleles). However, the clusters seen in the upper right quadrant of each plot resulted from signals from amplicons representing WTs (non-targeted mlo alleles).

Eighty six of the individual plants analyzed by ddPCR were further analyzed by PCR fragment size analysis to obtain additional information on RNP targeting of the mlo subgenomic A allele (primer combination Seq ID NO:1/Seq ID NO: 2). The product (2. Mu.l from 50. Mu.l total PCR volume) was isolated using Fragment AnalyzerTM systems (advanced analytical techniques Inc. (ADVANCED ANALYTICAL Technologies Inc.), AATI). The fragment analyzer is a silica-based Capillary Electrophoresis (CE) instrument with a gel matrix (DNF-920) and an intercalating dye and LED light source to easily quantify and quantify DNA fragments. Using3.0 Software (AATI) analyzes the raw data from Fragment AnalyzerTM to provide information about the size and concentration of the isolated DNA fragments. 6 plants from pool TMTA-035-B04, 25 plants from pool TMTA0233-037-B01, 14 plants from pool TMTA0233-037-B02, 16 plants from pool TMTA0233-044-B03 and 25 plants from pool TMTA0233-046-B02 were co-analyzed (FIG. 14). Among the individual plants from each analysis pool, a significant number of plants predicted to carry mlo mutations by ddPCR shed analysis showed variations in fragment length (typically with multiple fragments). In contrast, no multiple fragments were observed in the plants that showed 0% abscission during ddPCR analysis (27 plants marked with arrows). Interestingly, different fragment patterns were observed within the individual plants analyzed from a single pool, indicating that not all plants analyzed were cloned.

Twenty nine of the individual plants analyzed by PCR fragment size analysis were further analyzed by NGS to obtain precise sequence information for a and further B and D mlo subgenomic copies (table 4). Plants selected for NGS included 7 plants predicted by ddPCR that both a mlo subgenomic alleles were WTs to check for the presence of additional mutations on the B and D subgenomic copies. 500ng of purified PCR product (primer set Seq ID NO:11/Seq ID NO: 12) was sent to NGSGermany stock). amplicon-EZ Illumina based services provide full sequence coverage of PCR products up to 500bp in length (up to 50.000 reads/sample delivered). The raw Illumina data is analyzed and visualized using a software application CLC Genomics Workbench 12.0.3 (Qiagen) and proprietary CRISPRMAPPER plug-in. Overall, the results generally show a close correlation between the partition species of percentage of a subgenomic shedding and NGS, e.g., TMTA-032-B01-08 shows 0% shedding as determined by ddPCR, and confirmation by NGS that both a subgenomic mlo alleles are WTs; TMTA0233-035-B03-07 shows 52% shedding as determined by ddPCR and confirms by NGS that one of the mlo alleles carries a 5bp deletion and the other allele is WT (monoallelic); TMTA0233-047-B01-01 shows 100% shedding as determined by ddPCR, and here it is confirmed by NGS that one mlo allele carries a 2bp deletion and the other allele carries a 10bp deletion (bi-allele). For plants with no correlation between the assigned species of the subgenomic shedding percentage and NGS results, it was observed that at least one of the subgenomic alleles a carries a +1bp insertion, which may not be sufficient to prevent binding of the NHEJ shedding probe. NGS results also show that B and D subgenomic copies of mlo are also effectively targeted, with some plants carrying indels in all 6 mlo copies (e.g., TMTA-032-B01-06, TMTA-0233-035-B04-01, and TMTA-0233-044-B03-13). Given that the gRNA used has a 1bp mismatch with the target sequence, targeting of the B subgenomic allele of mlo is not expected to be so effective.

To provide data on the number of unique mutant plants that can be recovered from one bombarded immature embryo, 23 plants from explants TMTA-037 were analyzed by NGS: 7 plants were previously characterized as 50% ddPCR shed of the mlo subgenomic allele a, 7 plants were previously characterized as 100% ddPCR shed, 5 plants were previously characterized as 0% ddPCR shed, and 4 plants had a median percentage ddPCR shed. NGS results showed that 14 of the 23 plants selected from single embryo explants carried a unique mutation profile (table 5).

Analysis of offspring populations

To provide data on the genetic characteristics of Cas 9-induced mutations, 30 seeds from 3 self-pollinated plants (TMTA-033-032-B01-06, TMTA-035-B04-01 and TMTA-0233-044-B03-13) previously characterized as carrying 6 targeted mlo alleles were sown in the greenhouse, sampled and analyzed by NGS to determine indel delivery. All predicted mutations were recovered in the 3 offspring populations and isolated as expected (tables 11, 12 and 13).

EXAMPLE 2-mlo Cas12a RNP targeting

Cas12 a-targeted gRNA design for mlo

Based on previous work (Gil-Humanes et al 2017), we designed a synthetic crRNA targeting wheat mildew resistance locus O (mlo) consisting of a 21bp direct repeat and a 24bp pre-spacer. The mlo gene encodes a seven transmembrane domain protein which is involved in resistance to the fungal pathogen powdery mildew. The recognition site of crRNA is located within exon 4 of the antisense strand of mlo [5'- [ (TTTG) CGAACTGGTATTCCAAGGAGGCGG-3' ], with the PAM site between brackets. The designed guide was specific for the 5A and 4D alleles of mlo and showed a mismatch with the 4B allele at position 16 from the PAM sequence (fig. 15).

Microdroplet digital PCR (ddPCR) assay to identify Cas12a RNP-induced mlo insertion deletion mutations

The ddPCR assay for identifying Cas12a-RNP induced mlo indels is identical to the previously described ddPCR assay for detecting Cas9-RNP induced mlo indels (see Cas9 RNP targeting for ddPCR design, example 1-mlo). The predicted position of Cas12a cleavage site in the mlo gene, and the positions of PCR primers and probes for ddPCR analysis are shown in figure 16.

Wheat donor plant growth for immature embryo isolation

The donor plants of variety Fielder are grown under controlled environmental conditions to ensure optimal immature embryo quality. Typical growth conditions are day/night temperatures of 20 ℃ +/-1 ℃ and 18 ℃ +/-1 ℃, relative humidity of 65%, 16h photoperiod (400 μmol/m ²/s desktop level illuminance) provided by a mixture of 600W high pressure sodium lamp and 400W metal halide lamp.

Preparation of wheat immature embryos for bombardment

Embryos were aseptically excised from immature seeds using a binocular microscope (model MZ6, lycra company) and the hypocotyls passing through the green seed coats were carefully excised during preparation. Subsequent steps of embryo preparation were performed using the modified procedure substantially as described in Ishida et al (2015). Immature embryo explants were transferred to a 3.5cm petri dish (Fukai company 351008) containing 4.5ml of non-selective callus induction medium WLS (referred to herein as callus induction medium 240). The embryos are arranged in a 1.5cm center circle (25-50 embryos/petri dish, scutellum side up).

Preparation of Cas12a RNP complexes

Purified Cas12a nuclease and mlo specific crrnas were ordered from IDT (integrated DNA technologies) for RNP assembly.

-CrRNA: custom and user-defined crRNA that binds 24 bases on the DNA strand opposite TTTV (PAM sequence)

-Cas12a nuclease: (l.b. cas12a supernuclease): a recombinant trichomonadaceae (Lachnospiraceae) bacterial Cas12a nuclease purified from an escherichia coli strain expressing Cas12 a. Contains a C-terminal Nuclear Localization Signal (NLS) and a C-terminal 6-His tag.

For RNP complex assembly, mlo-specific crRNA and Cas12a nuclease were mixed in near equimolar ratio in NEBuffer ^TM 2.1.1 (new england biological laboratories inc.). The mixture was incubated at 37℃for 20-30min and then transferred to ice.

Cas12a RNP complex delivery to gene gun in wheat

Modified procedure use for Cas12a RNP delivery using Liang et al (2018)PDS-1000/He particle delivery System (Berle Corp.) delivers RNPs into immature embryos. RNP complex was combined with 0.6 μm gold particles (Berle Co.) andTransfection reagents (sigma-aldrich) were mixed, 15 μl aliquots were applied to the central region of each large carrier, and air dried in a laminar flow bench for 30min prior to delivery. Typically, for each shot, 150 μg of gold particles and 3 μg of Cas12a protein complexed with crRNA are delivered.

Cultivation of bombarded wheat immature embryos and plant regeneration

Following bombardment, immature embryos are incubated on the same plates in darkness (28 ℃ +/-1 ℃ C., 55% relative humidity) for 48H in an MLR-352H-PE Panasonic incubator. After this period, the embryos are transferred to fresh non-selective callus induction medium 240 (9 cm dishes containing 35-40ml medium, up to 15 embryos/dish) and incubated in the dark (25 ℃ +/-1 ℃). At this stage, embryos from different locations in the target region are separated (fig. 5), i.e. from the center region (region 1), the inner ring region (region 2) and the outer ring region (region 3).

Seven days after bombardment, immature embryos are split longitudinally into 2 pieces under a binocular microscope and transferred to fresh non-selective callus induction medium 240 (9 cm dishes, 12 embryos/dish containing 35-40ml medium) and cultured in the dark (25 ℃ +/-1 ℃).

Two weeks later, small pieces of structured embryogenic callus were transferred to non-selective regeneration medium LSZ (referred to herein as regeneration medium 420) (Ishida et al 2015). Regeneration medium 420 was prepared in PlantConTM ^TM containers (MP biomedical Co., catalog number 26-722-06), 100ml medium/container. Embryogenic callus produced from one embryo was transferred to one PlantCon ^TM container (up to 16 pieces per container). In case more than 16 embryogenic calli are recovered from one embryo, an additional regeneration vessel is utilized. The PlantCon ^TM vessels were incubated under light (23 ℃ +/-1 ℃ for 16h photoperiod) for approximately 6 weeks.

Shoots from PlantCon ^TM containers were transferred individually to De Wit tubes (Duchefa Biochemie company) containing 10ml of non-selective rooting medium WRM (essentially a modification of medium R) (Sparks and Jones, 2009), solidified with 0.15% w/v Gelrite (Duchefa Biochemie company) and cultured under light (23 ℃ +/-1 ℃ C., 16h photoperiod). The rooted plants were transferred to a greenhouse.

Tissue sampling and DNA isolation

Three days after sampling 1-bombardment, 5 immature embryos were randomly selected from region 2 (fig. 5) of each bombarded plate. Expanding the embryo as a sample, and collecting in a 2ml tubeSafe-Lock) and stored at-80 ℃. The samples were ground in a Retsch hybrid mill MM300/400 for 60s. Genomic DNA extraction was performed using QIAGEN DNEASY plant mini kit (catalog No. 69106) according to the manufacturer's instructions. The final DNA concentration was measured and the plates were stored at 4 ℃ until used for downstream analysis.

Sampling 2-leaves were harvested from regeneration shoots after culturing the structured embryogenic callus on non-selective regeneration medium 420 in PlantCon ^TM vessels for 4 weeks. Care was taken to ensure that similarly sized leaves were obtained from all regenerated shoots, which was best achieved by first cutting all longer tips with sterile scissors, and then cutting leaves of approximately 5mm length from the remaining tissue (fig. 6). The leaves from each PlantCon ^TM containers were combined and transferred to a 6ml screw cap tube (mei-ju MP company 32301) and stored at-80 ℃. Samples were freeze-dried overnight and genomic DNA was extracted using a LGC GENOMICS sbeadexTM Maxi plant kit based procedure with KingFisher automation. The final DNA concentration was measured and the plates were stored at 4 ℃ until used for downstream analysis.

Selection/analysis procedure to identify Cas12a RNP indels in mlo

FIG. 7 shows an overview of a 3-step selection system for identifying RNP-induced insert deletions in wheat plants mlo. This process involves sequential enrichment of tissues carrying RNP-induced mlo insertion deletions by repeated sampling and molecular screening to specifically detect targeted mutations. In the example shown, this is done in 3 steps in immature embryo explants, in a population of regenerated shoots, finally on a single plant level. A detailed overview of the selection/analysis process of Cas12a RNPs is shown in fig. 17 and 18.

Three days after gene gun RNP delivery, 5 immature embryos from each shot were selected from the inner loop region (region 2) and sampled for DNA isolation and ddPCR analysis to determine the percentage of mlo NHEJ shedding. The 20 XddPCR mixture consisted of 18. Mu.M forward primer (Seq ID NO: 1) and 18. Mu.M reverse primer (Seq ID NO: 2), 5. Mu.M reference probe (Seq ID NO: 3) and 5. Mu.M shedding probe (Seq ID NO: 5). The following reagents were mixed in 96-well plates to perform a 25 μl reaction: 11. Mu.l of probe ddPCR Supermix (without dUTP), 1.1. Mu.l of 10 Xassay mix (Berle laboratories Inc.), 100-250ng of genomic DNA in water, and water (up to 22. Mu.l). The results are summarized in table 6, with ddPCR shedding percentages ranging from 0.5% to 2.7%.

FIG. 19 shows a two-dimensional plot of immature embryo samples TMTA0527-B01$001, TMTA0527-B02$001, and TMTA0527-B05$001 analyzed by ddPCR generated by Quantasoft software. The cluster seen in the upper left quadrant of each panel resulted from signals from amplicons representing NHEJ indels (RNP-targeted mlo alleles). However, the clusters seen in the upper right quadrant of each plot resulted from signals from amplicons representing WTs (non-RNP targeted mlo alleles). Samples TMTA0527-B01$001, TMTA0527-B02$001, and TMTA0527-B05$00 showed the highest percent shedding relative to all bombarded plates, thus further culturing the remaining embryos from these shots alone. ddPCR results indicate that these immature embryos are the most likely to carry cells with NHEJ indels in the mlo allele.

Immature embryos are cultured on non-selective callus induction medium 240 for 3 cycles to obtain structured embryogenic callus from which plants can be regenerated in PlantCon ^TM vessels on non-selective regeneration medium 420. Leaves from multiple shoots in each PlantCon ^TM containers were pooled and transferred to a 6ml screw cap tube (michaustorium MP company 32301) for ddPCR analysis to determine the percentage of mlo shedding. From the 3 cultured shots of TMTA0527, a total of 139 pools from 46 individual embryos (58 pools from shot 1, 31 pools from shot 2, and 50 pools from shot 5) were analyzed. For ease of presentation, the mlo shedding percentages for each pool are categorized into different categories (< 10%, 10% -25%, 25% -50% and > 50%). The results are shown in Table 7. Although varying greatly, each shot contained a pool that exhibited a high percentage of shedding (e.g., 1:27.6% pool exhibited a percentage of shedding of >25%, 2:58% pool exhibited a percentage of shedding of >25%, and 5:8% pool exhibited a percentage of shedding of > 25%).

FIG. 20 shows a two-dimensional plot of representative pools generated by Quantasoft software showing the percentage of ddPCR shedding of <10% (TMTA 0527-031-B01 $001), 10% -25% (TMTA 0527-033-B02 $001), 25% -50% (TMTA 0527-039-B04 $001), and >50% (TMTA 0527-044-B06 $001). The cluster seen in the upper left quadrant of each panel resulted from signals from amplicons representing NHEJ indels (RNP-targeted mlo alleles). However, the clusters seen in the upper right quadrant of each plot resulted from signals from amplicons representing WTs (non-targeted mlo alleles).

Shoots from pools exhibiting a percentage of shedding of >25% were mainly selected for further culture (13 PlantCon ^TM containers from shoot 1, 8 PlantCon ^TM containers from shoot 2, 10 PlantCon ^TM containers from shoot 5). ddPCR results indicate that these pools are the most likely pools to carry shoots with NHEJ insertion deletions in the mlo allele. In addition, shoots from the other 7 pools that showed lower percent shedding (10% -25% species) were also selected.

Shoots from selected containers were transferred to individual De Wit tubes containing 10ml of non-selective rooting medium WRM for further development. Approximately 2 weeks after transfer, leaf samples from individual plants were taken for ddPCR analysis to determine the percentage of mlo shedding. Samples were taken from a total of 440 individual shoots derived from 24 immature embryo explants. Shoots were assigned to specific species based on the percentage of shedding obtained with ddPCR assays specifically designed for the a subgenomic group (i.e., near 0% value = WT, near 50% value = single allele mutation, and near 100% value = double allele mutation). The results of 436 shoots that can be unambiguously assigned to a particular species are shown (Table 8). For the other four plants, a median percentage shedding was observed and could not be accurately assigned to any species. Of 436 plants categorized into different categories, 224 plants (51%) showed a percentage of abscission consistent with mlo RNP targeting: 155 plants had a percentage of abscission of 50% (monoallelic), and 69 plants had a percentage of abscission of 100% (biallelic). FIG. 21 shows a two-dimensional map of representative plants generated by Quantasoft software for each ddPCR shed percentage species (0% shed: TMTA0527-049-B06-16, TMTA0527-051-B01-01, TMTA0527-054-B01-10, TMTA0527-058-B01-10;50% shed: TMTA0527-049-B06-03, TMTA0527-051-B01-09, TMTA0527-053-B04-02, TMTA0527-058-B01-06; and 100% shed: TMTA0527-049-B06-02, TMTA0527-050-B01-03, TMTA0527-051-B01-06, TMTA 0527-058-B01-03). The cluster seen in the upper left quadrant of each panel resulted from signals from amplicons representing NHEJ indels (RNP-targeted mlo alleles). However, the clusters seen in the upper right quadrant of each plot resulted from signals from amplicons representing WTs (non-targeted mlo alleles).

Individual plants analyzed by ddPCR were further analyzed by PCR fragment size analysis to obtain additional information on RNP targeting of the A subgenomic allele of mlo (primer combination Seq ID No:1/Seq ID No: 2). The product (2. Mu.l from 50. Mu.l total PCR volume) was isolated using Fragment AnalyzerTM systems (advanced analytical techniques, AATI). The fragment analyzer is a silica-based Capillary Electrophoresis (CE) instrument with a gel matrix (DNF-920) and an intercalating dye and LED light source to easily quantify and quantify DNA fragments. Using3.0 Software (AATI) analyzes raw data from Fragment AnalyzerTM to provide information about the size and concentration of isolated DNA fragments (fig. 22). Considerable variation in fragment length (typically with multiple fragments) was observed in plants recovered from each pool. Twenty one of the individual plants analyzed by PCR fragment size analysis was selected for NGS to obtain precise sequence information for a and further B and D mlo subgenomic copies (table 9). Plants selected for NGS included 4 plants predicted by ddPCR that both subgenomic mlo alleles were WTs to check for the presence of additional mutations on the B and D subgenomic copies. 500ng of purified PCR product (primer set Seq ID NO:11/Seq ID NO: 12) was sent to NGSGermany stock). amplicon-EZ Illumina based services provide full sequence coverage of PCR products up to 500bp in length (up to 50.000 reads/sample delivered). The raw Illumina data is analyzed and visualized using a software application CLC Genomics Workbench 12.0.3 (qiagen) and proprietary CRISPRMAPPER plug-in. Overall, the results generally show a close correlation between the partition species of percentage of a subgenomic shedding and NGS, e.g., TMTA-0527-049-B06-07 shows 0% shedding as determined by ddPCR, and confirmation by NGS that both a subgenomic mlo alleles are WTs; TMTA0527-049-B06-03 shows a 51% shedding as determined by ddPCR and confirms by NGS that one of the mlo alleles carries a10 bp deletion and the other allele is WT (single allele); TMTA0527-049-B06-17 shows 100% shedding as determined by ddPCR and here by NGS it is confirmed that one mlo allele carries a 3bp deletion and the other allele carries a 6bp deletion (bi-allele). For one plant analyzed that has no correlation between the assigned species of the A subgenomic shedding percentage and the NGS results, it was observed that one of the A subgenomic alleles carried a 1bp insert, which may not be sufficient to prevent binding of the NHEJ shedding probe (TMTA 0527-044-B06-16). NGS results also show that B and D subgenomic copies of mlo are also targeted, however targeting of the B subgenomic allele of mlo is less efficient, probably due to +1bp mismatch of Cas12a gRNA to the target sequence.

To provide data on the number of unique mutant plants that can be recovered from one bombarded immature embryo (TMTA 0527-045), 35 plants from 62 plants analyzed by PCR fragment size (fig. 23) were selected for NGS:15 plants were previously characterized as 50% ddPCR shed of the mlo subgenomic A allele, 15 plants were previously characterized as 100% ddPCR shed, and 5 plants were previously characterized as 0% ddPCR shed. Plants showing different segment patterns (varying length and number) were selected. NGS results showed that 32 of 35 plants selected from single embryo explants carried unique mutation profiles (table 10).

Example 3-Cas 12a RNP targeting of FLC

Cas12 a-targeted gRNA design for FLC

The Flowering Locus C (FLC) gene is known to play a key role in the vernalization response (the key process of regulating flowering time) of wheat. IWGSC REFSEQ V1.0.0 (Appels et al 2018) was used to design CRISPR-Cas12a RNP targeting methods for one of these FLC genes (TaAGL) present on all subgenomic groups (a (TraesCS a01G 435000), B (TraesCS 3B01G 470000) and D (TraesCS 3D01G 428000)). Synthetic Cas12a crrnas consisting of a 21bp co-repeat and a 23bp pre-spacer were designed to target all three TaAGL33 alleles. The recognition site for Cas12a crRNA is located on the sense strand of TaAGL '- [ (TTTC) AGCATAGAAGGTACATATGACCG-3' ], with the PAM site between brackets (fig. 24).

Microdroplet digital PCR (ddPCR) assay to identify Cas12a RNP-induced FLC insertion deletion

DdPCR assays were designed and validated to identify Cas12 a-RNP-induced FLC insertion deletions at the cr-FLC-G1 target site. The predicted position of Cas12a cleavage site in FLC gene, the positions of PCR primers and probes for ddPCR analysis are shown in fig. 25.

Wheat donor plant growth for immature embryo isolation

Preparation of wheat immature embryos for bombardment

Preparation of Cas12a RNP complexes

Purified Cas12a nuclease and FLC specific crRNA were ordered from IDT (integrated DNA technologies) for RNP assembly.

-CrRNA: custom and user-defined crRNA that binds to 23 bases on the DNA strand opposite TTTV (PAM sequence)

-Cas12a nuclease: (l.b. cas12a supernuclease): a recombinant trichomonadaceae bacterial Cas12a nuclease purified from an escherichia coli strain expressing Cas12 a. Contains a C-terminal Nuclear Localization Signal (NLS) and a C-terminal 6-His tag.

For RNP complex assembly, FLC specific crRNA and Cas12a nuclease were mixed in a near equimolar ratio in NEBuffer ^TM 2.1.1 (new england biological laboratories inc.). The mixture was incubated at 37℃for 20-30min and then transferred to ice.

Cas12a RNP complex delivery to gene gun in wheat

Cultivation of bombarded wheat immature embryos and plant regeneration

Following bombardment, immature embryos are incubated on the same plates in darkness (28 ℃ +/-1 ℃ C., 55% relative humidity) for 48H in an MLR-352H-PE Panasonic incubator. After this period, the embryos are transferred to fresh non-selective callus induction medium 240 (9 cm dishes containing 35-40ml medium, up to 15 embryos/dish) and incubated for an additional 1 day under the same conditions before being transferred to 25 ℃ (+/-1 ℃).

One week after bombardment, the immature embryos are split longitudinally into 2 pieces under a binocular microscope and transferred to fresh non-selective callus induction medium 240 (9 cm dishes, 12 embryos/dish containing 35-40ml medium) and cultured in the dark (25 ℃ +/-1 ℃).

Two weeks later, small pieces of structured embryogenic callus were transferred to non-selective regeneration medium LSZ (referred to herein as regeneration medium 420) (Ishida et al 2015). Regeneration medium 420 was prepared in PlantConTMTM containers (MP biomedical Co., catalog number 26-722-06), 100ml medium/container. Embryogenic callus produced from one embryo was transferred to one PlantConTM container (up to 16 pieces per container). In case more than 16 embryogenic calli are recovered from one embryo, an additional regeneration vessel is utilized. The PlantConTM vessels were incubated under light (23 ℃ +/-1 ℃ for 16h photoperiod) for approximately 6 weeks.

Shoots from PlantConTM containers were transferred individually to De Wit tubes (Duchefa Biochemie company) containing 10ml of non-selective rooting medium WRM (essentially a modification of medium R) (Sparks and Jones, 2009), solidified with 0.15% w/v Gelrite (Duchefa Biochemie company) and cultured under light (23 ℃ +/-1 ℃ C., 16h photoperiod).

Tissue sampling and DNA isolation

Sampling 2-leaves were harvested from regeneration shoots after culturing the structured embryogenic callus on non-selective regeneration medium 420 in PlantConTM vessels for 4-5 weeks. Care was taken to ensure that similarly sized leaves were obtained from all regenerated shoots, which was best achieved by first cutting all longer tips with sterile scissors, and then cutting leaves of approximately 5mm length from the remaining tissue (fig. 6). The leaves from each PlantConTM containers were combined and transferred to a 6ml screw cap tube (mei-ju MP company 32301) and stored at-80 ℃. Samples were freeze-dried overnight and genomic DNA was extracted using a LGC GENOMICS sbeadexTM Maxi plant kit based procedure with KingFisher automation. The final DNA concentration was measured and the plates were stored at 4 ℃ until used for downstream analysis.

Selection/analysis procedure to identify Cas12a RNP indels in FLCs

FIG. 7 shows an overview of a 3-step selection system for identifying RNP-induced insert deletions in wheat plant FLCs. This process involves sequential enrichment of tissues carrying RNP-induced FLC insertion deletions by repeated sampling and molecular screening to specifically detect targeted mutations. In the example shown, this is done in 3 steps in immature embryo explants, in a population of regenerated shoots, finally on a single plant level. A detailed overview of the selection/analysis process of Cas12a RNPs is shown in fig. 17 and 18.

Three days after gene gun RNP delivery, 5 immature embryos from each shot were selected from the inner loop region (region 2) and sampled for DNA isolation and ddPCR analysis to determine FLC NHEJ shedding percentages. The 20 Xassay mixture consisted of 18. Mu.M forward primer (Seq ID NO: 13) and 18. Mu.M reverse primer (Seq ID NO: 14), 5. Mu.M reference probe (Seq ID NO: 15) and 5. Mu.M shedding probe (Seq ID NO: 16). The following reagents were mixed in 96-well plates to perform a 25 μl reaction: mu.l of probe ddPCR Supermix (dUTP free, burley laboratories) 1.1. Mu.l of 20 Xassay mix, 100-250ng of genomic DNA in water, and water (up to 22. Mu.l). The results are summarized in Table 14, with ddPCR shedding percentages ranging from 0.91% to 6.87%. Samples from shots 2, 3 and 4 showed the highest percent shedding, and therefore the remaining embryos from these shots were further cultured. ddPCR results indicate that these immature embryos are the most likely to carry cells with NHEJ indels in the FLC allele.

Immature embryos are cultured on non-selective callus induction medium 240 for 3 cycles to obtain structured embryogenic callus from which plants can be regenerated in PlantConTM vessels on non-selective regeneration medium 420. Leaves from multiple shoots in each PlantConTM containers were pooled and transferred to a 6ml screw cap tube (michaux MP company 32301) for ddPCR analysis to determine the percentage of FLC shedding. From the TMTA0609 3-cultured shots, a total of 156 pools from 79 individual embryos (59 pools from shot 2, 37 pools from shot 3, and 60 pools from shot 4) were analyzed. For ease of presentation, the percentage of FLC shedding for each pool was categorized into different categories (+.ltoreq.10%, >10% +.ltoreq.25%, >25% +.ltoreq.50% and > 50%), with the results shown in Table 15. As expected, shoot 3, which showed the highest percent shedding at sampling step 1 (immature embryo), exhibited a higher shedding value in the pooled leaf samples, with about 30% of the pools having a shedding value of > 25%.

Shoots from pools exhibiting the highest percent shedding (10 pools from shoot 2, 16 pools from shoot 3 and 8 pools from shoot 4) were selected for further culture. ddPCR results indicate that these pools are the most likely pools to carry shoots with NHEJ insertion deletions in the FLC allele.

Shoots from selected containers were transferred to individual De Wit tubes containing 10ml of non-selective rooting medium WRM for further development. Approximately 2 weeks after transfer, leaf samples from individual plants were taken for ddPCR analysis to determine the percentage of mlo shedding. Samples were taken from 605 individual shoots derived from 32 immature embryo explants altogether. Based on the percentage of shedding predicting the number of targeted alleles, shoots are assigned to specific FLC targeted species. Since ddPCR assays can detect insertion deletions on all subgenomic copies of FLC, the percent shedding can directly estimate the number of targeted alleles in each plant; About 17% abscission = 1 targeted allele, about 33% abscission = 2 targeted alleles, about 50% abscission = 3 targeted alleles, about 67% abscission = 4 targeted alleles, about 83% abscission = 5 targeted alleles, and 100% abscission = all 6 targeted alleles. The results of 589 shoots that can be unambiguously assigned to a particular species are shown (Table 16). For the other 16 plants, ddPCR failed, or the plants could not be assigned to any species accurately. For shoot 2, 189 plants were categorized into various categories (32 plants with one targeted allele, 18 plants with 2 targeted alleles, 28 plants with 3 targeted alleles, 8 plants with 4 targeted alleles, 2 plants with 5 targeted alleles, 2 plants with all 6 targeted alleles, and 99 plants without targeted alleles). Of the 138 plants analyzed from shoot 4, 21 plants were identified with one targeted allele, 12 plants with 2 targeted alleles, 15 plants with 3 targeted alleles, 16 plants with 4 targeted alleles, 5 plants with 5 targeted alleles, 11 plants with all 6 targeted alleles, and 58 plants without targeted alleles. For shoot 3, 262 plants were categorized into various categories (17 plants had one targeted allele, 35 plants had 2 targeted alleles, 27 plants had 3 targeted alleles, 25 plants had 4 targeted alleles, 35 plants had 5 targeted alleles, 32 plants had all 6 targeted alleles, and 91 plants had no targeted alleles). Most mutant plants were identified from shoot 3, as expected, because the highest percentage of abscission was also observed in 2 previous sampling steps (immature embryos and pooled leaf samples). Furthermore, the total number of FLC targeting alleles in each plant was also more prominent in shoot 3, where the multiallelic targeting levels on the A, B and D copies of FLC were higher (fig. 26). None of the FLC-targeted plants were further characterized by NGS to accurately define the insertion deletion at the sequence level. Thus, there is a possibility that some plants recovered in the same pool and belonging to the same FLC targeting species are clones with the same mutation profile.

Reference to the literature

Appels R,et al.Shifting the limits in wheat research and breeding using a fully annotated reference genome.Science,2018,361(6403):361–374.

Gil-Humanes J,Wang Y,Liang Z,Shan Q,Ozuna CV,Sánchez-León S,Baltes NJ,Starker C,Barro F,Gao C,Voytas DF.High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.Plant J.2017Mar;89(6):1251-1262.

Ishida Y,Tsunashima M,Hiei Y,Komari T.Wheat(Triticum aestivum L.)transformation using immature embryos.Methods Mol Biol.2015;1223:189-98.

Liang Z,Chen K,Zhang Y,Liu J,Yin K,Qiu JL,Gao C.Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins.Nat Protoc.2018Mar;13(3):413-430.

Sparks CA,Jones HD.Biolistics transformation of wheat.In:Jones HD,Shewry PR,editors.Transgenic wheat,barley and oats:production andcharacterization protocols.Totowa:Humana Press;2009.p.71-92.

Claims

1. A method for producing a plant comprising a desired nucleic acid sequence from a population of plant cells comprising regenerative cells, the population of plant cells comprising a subpopulation of cells comprising the desired nucleic acid sequence, wherein the method comprises the steps of:

a) Dividing a plant cell population comprising regenerative cells into subgroups, the plant cell population comprising a subpopulation of cells comprising a desired nucleic acid sequence, quantifying the concentration of the desired nucleic acid sequence for each subgroup, each subgroup representing a subset of the genotype of the population, and identifying one or more subgroups having the highest concentration of the desired nucleic acid sequence,

B) Culturing cells from the one or more subsets having the highest concentration of the desired nucleic acid sequence, dividing the cells into subsets, quantifying the concentration of the desired nucleic acid sequence in each subset, each subset representing a subset of the population genotype, and selecting the one or more subsets having the highest concentration of the desired nucleic acid sequence, and

2. The method of claim 1, wherein the regenerative cells are selected from the group consisting of:

a. Single cells, such as protoplasts or microspores,

B. cell aggregates, such as cell suspensions or callus cultures,

C. Complex multicellular explants from mature or immature seeds, such as immature embryos, scutellum or cotyledons,

D. complex multicellular explants from seedlings, e.g., root, hypocotyl, cotyledon, leaf, petiole or meristem, and

E. Complex multicellular explants from plants, such as roots, leaves, leaf bases, petioles, stems or meristems.

3. The method of claim 1 or 2, wherein the population of plant cells comprising regenerative cells is first divided into subgroups and then the concentration of the desired nucleic acid sequence in said subgroups in each subgroup is tested.

4. The method of any one of claims 1 to 3, comprising the step of chemically mutating or ionizing, gene or genome editing or gene engineering the nucleic acid molecules or genomes of the plant cells prior to dividing the cells into subgroups.

5. The method of any one of claims 1 to 3, comprising the step of chemically mutating or ionizing, gene or genome editing or gene engineering the nucleic acid molecules or genomes of the plant cells after a subset of the regenerated cells has been formed.

6. The method of any one of claims 1 to 5, wherein the medium or growth conditions in which the cells, tissues or plants are cultured are not selective for the presence of the desired nucleic acid sequence in the genome of the regenerating cells.

7. The method of any one of claims 1 to 6, comprising the step of extracting the nucleic acid molecules from said sample of each subset of genetically modified cells and destroying cells of the sample when analyzing the concentration of the desired nucleic acid sequence in the sample.

8. The method of any one of claims 1 to 7, comprising determining the concentration of the nucleic acid sequence in the genome of the genetically modified plant cells in molecular sieves, preferably by NGS or ddPCR.

9. The method of any one of claims 1 to 8, comprising the steps of: (i) culturing the one or more selected subsets of cells from step (a), (ii) extracting nucleic acid molecules from the cells from one or more samples from each subset, (iii) identifying one or more subsets having the highest concentration of the desired nucleic acid sequence, and (iv) selecting one or more subsets of the cells having the highest concentration of the desired nucleic acid sequence.

10. The method according to any one of claims 1 to 9, comprising the steps of: (i) recovering individual plants from the cells of the one or more selected subgroups from step (b), (ii) dividing the population of plants into subgroups, (ii) extracting DNA from one or more samples of said plants from each subgroup, (iii) combining samples taken from individual plants from a subgroup and identifying one or more subgroups having the highest concentration of the desired nucleic acid sequence(s), and (iv) selecting the one or more subgroups of said plants having the highest concentration of the desired nucleic acid sequence(s).

11. A method for producing a plant comprising a desired nucleic acid sequence, the method comprising the steps of:

(a1) (i) providing a population of plant cells comprising regenerated plant cells expected to have a desired nucleic acid sequence, (ii) dividing the population of plant cells comprising regenerated plant cells into subgroups, (iii) extracting DNA from one or more samples of said cells from subgroups, (iv) identifying one or more subgroups having one or more highest concentrations of the desired nucleic acid sequence, and (v) selecting one or more subgroups of said cells having the highest concentrations of the desired nucleic acid sequence,

Or alternatively

(A2) (i) dividing a population of plant cells comprising regenerated plant cells into subgroups, (ii) optionally, genetically modifying the subgroups of plant cells comprising regenerated plant cells, (iii) extracting DNA from one or more samples of said cells from each subgroup, (iv) identifying one or more subgroups having one or more highest concentrations of a desired nucleic acid sequence, and (v) selecting one or more subgroups of said cells having one or more highest concentrations of the desired nucleic acid sequence,

And

(B) (i) culturing the one or more selected subsets of regenerated plant cells of step (a 1) or (a 2), (ii) extracting DNA from one or more samples of cultured plant cells from these subsets, (iv) identifying one or more subsets having one or more highest concentrations of the desired nucleic acid sequence, and (v) selecting one or more subsets of the cultured cells having one or more highest concentrations of the desired nucleic acid sequence,

And

(C1) (i) recovering individual plants or individual shoots from the one or more selected subsets of cells from step (b), (ii) dividing the plant or shoot population into subsets, (ii) extracting DNA and/or RNA from one or more samples from the cells of each subset, (iii) identifying one or more subsets having the highest concentration of the desired nucleic acid sequences, and (iv) selecting and growing one or more subsets of the plants or shoots having the highest concentration of the desired nucleic acid sequences,

Or alternatively

(C2) (i) recovering individual plants or individual shoots from the cultured cells from the one or more selected subsets of step (b), (ii) obtaining a sample comprising DNA and/or RNA from each plant or each shoot, (iii) analysing said DNA and/or RNA for the presence of the desired nucleic acid sequence, (iv) selecting plants having the desired nucleic acid sequence and growing the plants,

Wherein, optionally, these plant cells are genetically modified cells, preferably regenerated plant cells, e.g. cells produced by particle bombardment or agrobacterium transformation or protoplast transfection.